Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent

ABSTRACT

A multifunctional molecular complex for the transfer of a nucleic acid composition to a target cell is provided which comprises in any functional combination: A) said nucleic acid composition; and B) a transfer moiety comprising 1) one or more cationic polyamine components bound to said nucleic acid composition, each comprising from three to twelve nitrogen atoms; 2) one or more endosome membrane disruption promoting components attached to at least one nitrogen atom of at least one of said polyamine components, through an alkyl, carboxamide, carbamate, thiocarbamate, or carbamoyl bridging group, comprising a) at least one lipophilic long chain alkyl group, b) a fusogenic peptide comprising spike glycoproteins of enveloped animal viruses, or c) cholic acid or cholesteryl or derivatives; and optionally 3) one or more receptor specific binding components which are ligands for natural receptors of said target cell, attached through an alkyl, carboxamide, carbamate, thiocarbamate, or carbamoyl bridging group to either i) a further nitrogen atom of at least one of said polyamine components to which said one or more endosome membrane disruption promoting components is attached, or ii) a nitrogen atom of at least one further polyamine component which does not have attached thereto any endosome membrane disruption promoting component. Also provided are the transfer moiety alone, or in combination with the nucleic acid composition as a self-assembling combination, and the use of these compositions in methods for transfering nucleic acid compositions to cells or to cells of individuals, for immunizing individuals against a pathogen or disease, and for treating an individual with a disease.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is in the field of methods for the transfer ofgenetic information, e.g., foreign DNA, into target cells, especiallyeukaryotic cells. In particular, the present invention relates tononviral gene carriers comprising multifunctional molecular conjugateswhich include, inter alia, lipopolyamines of a particular configuration,a component which promotes endosome disruption, and a receptor specificbinding component.

Heretofore, viral vectors of various types have been successfullyutilized for the insertion of selected foreign genetic information intoa target cell, and in the case of eukaryotic cells, for incorporation ofthat genetic information into the genome of the cell. These viral vectorsystems have relied upon the molecular machinery of the virus, evolvedover time to surmount the significant problems facing a virus inattempting to invade, i.e., infect a cell. Despite the efficiency ofsuch viral vectors, however, there has been continued concern regardingthe safety of using viruses, particularly from the standpoint ofundesired side effects. Thus, there has been an ongoing effort todevelop non-viral gene delivery systems that are as efficient as viralvectors, but with an improved safety profile.

Nonviral vectors or carriers, of the type with which the presentinvention is concerned, will thus have to overcome the same obstacles asa viral vector. The problems faced by such carriers include persistencein the biophase of the organism for a sufficient time to reach thetarget cell; recognition of the target cell and means for mediatingtransport of the genetic material through the cell membrane and into thecytoplasm of the cell; avoidance of degradation within the cell by thereticuloendothelial system; and transport to and through the nuclearmembrane into the nucleus of the cell where transcription of the geneticmaterial can take place.

It is to overcoming the problems described above that the presentinvention is addressed; and since the problems are several anddifferent, the present invention comprises a multifunctional complex,i.e., a molecular conjugate of various ligands intended to surmountspecific obstacles.

The ultimate usefulness of gene transfer techniques is of enormouspotential benefit in a number of areas. The transfer of genetic materialinto cells is the basis of a number of processes now widely accepted inthe areas of molecular biology, gene therapy and genetic immunization.Transfer of the genetic information encoded in DNA to cells where itexpresses identified individual proteins, has permitted investigation ofthe function of such proteins on a cellular level, and of the underlyingcell physiology. Genetic material has also been transferred into cellsto introduce proteins that are absent due to an inherent genetic flaw inthe cell that expresses an inactive protein or else prevents expressionof the protein altogether. The transfer of genetic material into cellscan be used to prevent the expression of proteins in those cells throughthe well-known antisense effect of complementary DNA or RNA strands.

Exogenous, i.e., foreign genetic material can permit cells to synthesizesignificant amounts of proteins that are not available by other means inpractical economic terms. These proteins of interest can be grown in avariety of host cells such as yeast, bacterial or mammalian cells.Genetic material can also be used to provide protective immune responsesin vivo by injection of DNA that encodes immunogenic proteins, i.e.,ones that can stimulate the desired immune response. The in vivointroduction of exogenous genetic material into cells also has potentialutility in applications for the alleviation, treatment or prevention ofmetabolic, tumoral or infectious disorders by the same mechanismsenumerated above.

Description of the Prior Art

It is possible to transfer genetic material into target cells withoutthe use of vectors or carriers. For example, genetic material can beintroduced systemically through an intravenous or intraperitonealinjection for in vivo applications, or can be introduced to the site ofaction by direct injection into that area. For example, it has long beenrecognized that DNA, by itself, injected into various tissues, willenter cells and produce a protein that: will elicit an immune response.See, e.g., P. Atanasiu et al., Academie des Sciences (Paris) 254,4228-30 (1962); M. A. Israel et al., J. Virol. 29, 990-96 (1979); H.Will et al., Nature, 299, 740-42 (1982); H. Robinson, World PatentApplication WO 86/00930, published 13 Feb. 1986; P. L. Felgner, J. A.Wolff, G. H. Rhodes, R. W. Malone and D. A. Carson, World PatentApplication WO 90/11092, published 4 Oct. 1990; and R. J. Debs and N.Zhu, World Patent Application WO 93/24640 published 9 Dec. 1993.However, DNA by itself is hydrophilic, and the hydrophobic character ofthe cellular membrane poses a significant barrier to the transfer of DNAacross it. Accordingly, it has become preferred in the art to usefacilitators that enhance the transfer of DNA into cells on directinjection.

Another approach in the art to delivery of genetic material to targetcells is one that takes advantage of natural receptor-mediatedendocytosis pathways that exist in such cells. Several cellularreceptors have been identified heretofore as desirable agents by meansof which it is possible to achieve the specific targeting of drugs, andespecially macromolecules and molecular conjugates serving as carriersof genetic material of the type with which the present invention isconcerned. These cellular receptors allow for specific targeting byvirtue of being localized to a particular tissue or by having anenhanced avidity for, or activity in a particular tissue. See, e.g., J.L. Bodmer and R. T. Dean, Meth. Enzymol., 112, 298-306 (1985). Thisaffords the advantages of lower doses or significantly fewer undesirableside effects.

One of the better known examples of a cell and tissue selective receptoris the asialoglycoprotein receptor present in hepatocytes. Theasialoglycoprotein receptor is an extracellular receptor with a highaffinity for galactose, especially tri-antennary oligosaccharides, i.e.,those with three somewhat extended chains or spacer arms having terminalgalactose residues; see, e.g., H. F. Lodish, TIBS, 16, 374-77 (1991).This high affinity receptor is localized to hepatocytes and is notpresent in Kupffer cells; allowing for a high degree of selectivity indelivery to the liver.

It has also been proposed in the art of receptor-mediated gene transferthat in order for the process to be efficient in vivo, the assembly ofthe DNA complex should result in condensation of the DNA to a sizesuitable for uptake via an endocytic pathway. See, e.g., J. C. Perales,T. Ferkol, H. Beegen, O. D. Ratnoff, and R. W. Hanson, Proc. Nat. Acad.Sci. USA, 91, 4086-4090 (1994).

An alternative method of providing cell-selective binding is to attachan entity with an ability to bind to the cell type of interest; commonlyused in this respect are antibodies which can bind to specific proteinspresent in the cellular membranes or outer regions of the target cells.Alternative receptors have also been recognized as useful infacilitating the transport of macromolecules, such as biotin and folatereceptors; see P. S. Low, M. A. Horn and P. F. Heinstein, World PatentApplication WO 90/12095, published 18 Oct. 1990; P. S. Low, M. A. Hornand P. F. Heinstein, World Patent Application WO 90/12096, published 18Oct. 1990; P. S. Low, M. A. Horn and P. F. Heinstein, U.S. Pat. No.5,108,921, Apr. 28, 1992; C. P. Leamon and P. S. Low, Proc. Nat. Acad.Sci. USA, 88, 5572-5576 (1991); transferrin receptors; insulinreceptors; and mannose receptors (see further below). The enumeratedreceptors are merely representative, and other examples will readilycome to the mind of the artisan.

The conjugation of different functionalities on the same molecule hasalso been utilized in the art. For example, in 1988 G. Y. Wu and C. M.Wu, J. Biol. Chem., 263, 14621-14624 (1988) described a method forcellular receptor mediated delivery of DNA to hepatocytes. This methodwas further described in G. Y. Wu and C. H. Wu, Biochem., 27, 887-892(1988); G. Y. Wu and C. H. Wu, U.S. Pat. No. 5,166,320, Nov. 24, 1992;and G. Y. Wu and C. H. Wu, World Patent Application 1O 92/06180,published 16 Apr. 1992. The method consists of attaching a glycoprotein,asialoorosomucoid, to poly-lysines to provide a hepatocyte selective DNAcarrier. The function of the poly-lysine is to bind to the DNA throughionic interactions between the positively charged (cationic) ε aminogroups of the lysines and the negatively charged (anionic) phosphategroups of the DNA. Orosomucoid is a glycoprotein which is normallypresent in human serum. Removal of the terminal sialic acid (N-acetylneuraminic acid) from the branched oligosaccharides exposes terminalgalactose oligosaccharides, for which hepatocyte receptors have a highaffinity, as already described.

After binding to the asialoglycoprotein receptor on hepatocytes, theprotein is taken into the cell by endocytosis into a pre-lysosomalendosome. The DNA, ionically bound to the poly-lysine-asialoorosomucoidcarrier, is also taken into the endosome. Additional work using thisdelivery system, e.g., that done by J. M. Wilson, M. Grossman, J. A.Cabrera, C. H. Wu and G. Y. Wu, J. Biol. Chem, 267, 11483-11489 (1992),has found that partial hepatectomy improves the persistence of theexpression of the DNA delivered into the hepatocytes. The transfer ofthe DNA into cells by this mechanism is also significantly enhanced bythe addition of cationic lipids; see, e.g., K. D. Mack, R. Walzem and J.B. Zeldis, Am. J. Med. Sci., 307, 138-143 (1994).

The use of a specific asialoglycoprotein is not required to achievebinding to the asialoglycoprotein receptor; this binding can also beaccomplished with high affinity by the use of small, synthetic moleculeshaving a similar configuration. The carbohydrate portion can be removedfrom an appropriate glycoprotein and be conjugated to othermacromolecules; see, e.g., S. J. Wood and R. Wetzel, Bioconj. Chem., 3,391-396 (1992). By this procedure the cellular receptor binding portionof the glycoprotein is removed, and the specific portion required forselective cellular binding can be transferred to another molecule.

There is a ample literature on the preparation of synthetic glycosideswhich can be attached to macromolecules and confer on them the abilityto bind to the corresponding galactose specific receptor. The importanceof branched glycosides was recognized early; see Y. C. Lee, Carb. Res.,67, 509-514 (1978). Further work delineated that sugar density K.Kawaguchi, M. Kuhlenschmidt, S. Roseman and Y. C. Lee, J. Biol. Chem.,256,2230-2234 (1981)! and spacial relationships Y. C. Lee, R. R.Townsend, M. R. Hardy, J. Lonngrer, J. Arnarp, M. Haraldsson and H.Lonn, J. Biol. Chem., 258, 199-202 (1983)! are important determinants ofbinding potency. Reductive amination of a peptide with a branchedtri-lysine amino terminus gives a ligand ending with four galactosylresidues that can be readily coupled to poly-lysine or othermacromolecules; see C. Plank, K. Zatlouhal, M. Cotten, K. Mechtler andE. Wagner, Bioconj. Chem., 3,533-539(1992); and has been used to prepareDNA constructs.

Thiopropionate and thiohexanoate glycosidic derivatives of galactosehave been prepared and linked to L-lysyl-L-lysine to form a synthetictri-antennary galactose derivative. A bisacridine spermidine derivativecontaining this synthetic tri-antennary galactose has been used totarget DNA to hepatocytes; see F. C. Szoka, Jr. and J. Haensle, WorldPat Application WO 93/19768, published 14 Oct. 1993; and J. Haensler andF. C. Szoka, Jr., Bioconj. Chem., 4, 85-93 (1993).

Other means of providing cellular receptor based facilitation of genetransfer into cells using poly-lysine as a carrier have been describedin the art. Antibodies specific for cell surface thrombomodulin havebeen used with poly-lysine as a delivery system for DNA in vitro and invivo; see V. S. Trubetskoy, V. P. Torchilin, S. J. Kennel and L. Huang,Bioconj. Chem., 3, 323-327 (1992). The transferrin receptor has alsobeen used to target DNA to erythroblasts. K562 macrophages and ML-60leukemic cells; see E. Wagner, M. Zenke, M. Cotten, H. Beug and M. L.Birnstiel, Proc. Nat. Acad. Sci. USA, 87, 3410-3414 (1990); M. Zenke, P.Steinlein, E. Wagner, M. Cotten, H. Beug and M. L. Birnstiel, Proc. Nat.Acad. Sci. USA, 87, 3655-3659 (1990); and G. Citro, D. Perrotti, C.Cucco, I. D'Agnano, A. Sacchi, G. Zupi and B. Calabretta, Proc. Nat.Acad. Sci. USA, 89, 7031-7035 (1990). These studies used both smalloliogodeoxynucleotides as well as large plasmids.

The ability of poly-lysine to facilitate DNA entry into cells issignificantly enhanced if the poly-lysine is chemically modified withhydrophobic appendages; see X. Zhou and L. Huang, Biochim. Biophys.Acta, 1189, 195-203 (1994); complexed with cationic lipids; see K. D.Mack, R. Walzem and J. B. Zeldis, Am. J. Med. Sci., 307, 138-143 (1994)or associated with viruses. Many viruses infect specific cells byreceptor mediated binding and insertion of the viral DNA/RNA into thecell; and thus this action of the virus is similar to the facilitatedentry of DNA described above.

Replication-incompetent adenovirus has been used to enhance the entry oftransferrin-poly-lysine complexed DNA into cells; see D. T. Curiel, S.Agarwal, E. Wagner and M. Cotten, Proc. Nat. Acad. Sci. USA, 88,8850-8854 (1991); E. Wagner, K. Zatloukal, M. Cotten, H. Kirlappos, K.Mechtler, D. T. Curiel and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA,89, 6099-6103 (1992); M. Cotten, E. Wagner, K. Zatloukal, S. Phillips,D. T. Curiel and M. L. Birnstiel, Proc. Nat. Acad. Sci. USA, 89,6094-6098 (1992); and L. Gao, E. Wagner, M. Cotten, S. Agarwal, C.Harris, M. Romer, L. Miller, P. C. Hu and D. Curiel, Hum. Gene Ther., 4,17-24 (1993). The adenovirus enhances the entry of thepoly-lysine-transferrin-DNA complex when covalently attached to thepoly-lysine and when attached through an antibody binding site. Theredoes not need to be a direct attachment of the adenovirus to thepoly-lysine-transferrin-DNA complex, and it can facilitate the entry ofthe complex when present as a simple mixture. The poly-lysinetransferrin-DNA complex provides receptor specific binding to the cellsand is internalized into endosomes along with the DNA. Once inside theendosomes, the adenovirus facilitates entry of theDNA/transferrin-poly-lysine complex into the cell by disruption of theendosomal compartment with subsequent release of the DNA into thecytoplasm. Replication-incompetent adenovirus has also been used toenhance the entry of uncomplexed DNA plasmids into cells without thebenefit of the cell receptor selectivity conferred by thepoly-lysine-transferrin complex; see K. Yoshimura, M. A. Rosenfeld, P.Seth and R. G. Crystal, J. Biol. Chem., 268, 2300-2303 (1993).

Synthetic peptides such as the N-terminus region of the influenzahemagglutinin protein are known to destabilize membranes and are knownas fusogenic peptides. Conjugates containing the influenza fusogenicpeptide coupled to poly-lysine together with a peptide having a branchedtri-lysine amino terminus ligand ending with four galactosyl residueshave been prepared as facilitators of DNA entry into hepatocytes; see C.Plank, K. Zatlouhal, M. Cotten, K. Mechtler and E. Wagner, Bioconj.Chem., 3,533-539 (1992). These conjugates combine the asialoglycoproteinreceptor mediated binding conferred by the tetra-galactose peptide, theendosomal disrupting abilities of the influenza fusogenic peptide, andthe DNA binding of the poly-lysine. These conjugates deliver DNA intothe cell by a combination of receptor mediated uptake andinternalization into endosomes. This internalization is followed bydisruption of the endosomes by the influenza fusogenic peptide torelease the DNA into the cytoplasm. In a similar fashion, the influenzafusogenic peptide can be attached to poly-lysine and mixed with thetransferrin-poly-lysine complex to provide a similar DNA carrierselective for cells carrying the transferrin receptor; see E. Wagner, C.Plank, K. Zatloukal, M. Cotten and M. L. Birnstiel, Proc. Nat. Acad.Sci. USA, 89, 7934-7938 (1992). Synthetically designed peptides can alsobe used; for example the "GALA" peptides N. K. Subbarao, R. A. Parente,F. C. Szoka, Jr, L. Nadasdi and K. Pongracz, J. Biol. Chem., 26,2964-2972 (1987)! have been coupled to DNA carriers and an enhancedfacilitated entry into cells was observed J. Haensler and F. C. Szoka,Jr., Bioconj. Chem., 4, 372-379 (1993)!. The cationic amphipathicpeptide gramicidin S can facilitate entry of DNA into cells J. Y.Legendre and F. C. Szoka, Jr., Proc. Nat. Acad. Sci. USA, 90, 893-897(1993)!, but also requires a phospholipid to achieve significanttransfer of DNA.

Poly-lysine is not unique in providing a polycationic framework for theentry of DNA into cells. DEAE-dextran has also been shown to beeffective in promoting RNA and DNA entry into cells; see R. Juliano andE. Mayhew, Exp. Cell. Res. 73, 3-12 (1972); and E. Mayhew and R.Juliano, Exp. Cell. Res. 77, 409-414 (1973). More recently, a dendriticcascade co-polymer of ethylenediamine and methyl acrylate has been shownto be useful in providing a carrier of DNA which facilitates entry intocells; see J. Haensler and F. C. Szoka, Jr., Bioconj. Chem., 4, 372-379(1993). An alkylated polyvinylpyridine polymer has also been used tofacilitate DNA entry into cells; see A. V. Kabanov, I. V. Astafieva, I.V. Maksimova, E. M. Lukanidin, G. P. Georgiev and V. A. Kabanov,Bioconj. Chem., 4, 448-454 (1993).

Positively charged liposomes have also been widely used as carriers ofDNA which facilitate entry into cells; see, e.g., F. C. Szoka, Jr. andJ. Haensler, World Pat Application WO 93/19768, published 14 Oct. 1993;R. J. Debs and N. Zhu, World Patent Application WO 93/24640, published9Dec. 1993; P. L. Felgner, R. Kumar, C. Basava, R. C. Border and J. Y.Hwang-Felgner, World Patent Application WO 91/16024, published 31 Oct.1991; P. L. Felgner and G. M. Ringold, Nature, 337, 387-388 (1989); J.K. Rose, L. Buonocore and M. A. Whitt, BioTechniques, 10, 520-525(1991); C. F. Bennett, M. Y. Chiang, H. Chan, J. E. E. Schoemaker and C.K. Mirabelli, Mol. Pharm. 41, 1023-1033 (1992); J. H. Felgner, R. Kumar,C. N. Sridhar, C. J. Wheeler, Y. J. Tsai, R. Border, P. Ramsey, M.Martin and P. L. Felgner, J. Biol. Chem., 269, 2550-2561 (1994); J. G.Smith, R. L. Walzem and J. B. German, Biochim. Biophys. Acta, 1154,327-340 (1993). These carrier compositions have also included pHsensitive liposomes; see C. J. Chu, J. Dijkstra, M. Z. Lai, K. Hong andF. C. Szoka, Jr., Pharm. Res., 7, 824-854 (1990); J. Y. Legendres and F.C. Szoka, Jr., Pharm. Res., 9, 1253-1242 (1992).

A poly-cationic lipid has been prepared by couplingdioctadecylamidoglycine and dipalmitoyl phosphatidylethanolamine to a5-carboxyspermine; see J. P. Behr, B. Demeniex, J. P. Loeffler and J.Perez-Mutul, Proc. Nat. Acad. Sci. USA, 86, 6982-6986 (1989); F.Barthel, J. S. Remy, J. P. Loeffler and J. P. Behr, DNA and Cell Biol.,12, 553-560 (1993); J. P. Loeffler and J. P. Behr, Meth. Enzymol., 217,599-618 (1993); J. P. Behr and J. P. Loeffler, U.S. Pat. No. 5,171,678,Dec. 15, 1992. These lipophilic-spermines are very active intransferring DNA through cellular membranes.

Combinations of lipids have been used to facilitate the transfer ofnucleic acids into cells. For example, in U.S. Pat. No. 5,283,185 thereis disclosed such a method which utilizes a mixed lipid dispersion of acationic lipid with a co-lipid in a suitable solvent. The lipid has astructure which includes a lipophilic group derived from chlolesterol, alinker bond, a linear alkyl spacer arm, and a cationic amino group; andthe co-lipid is phosphatidylcholine or phosphatidylethanolamine.

Macrophages have receptors for both mannose and mannose-6-phosphatewhich can bind to and internalize molecules displaying these sugars. Themolecules are internalized by endocytosis into a pre-lysosomal endosome.This internalization has been used to enhance entry of oligonucleotidesinto macrophages using bovine serum albumin modified withmannose-6-phosphate and linked to an oligodeoxynucleotide by a disulfidebridge to a modified 3' end; see E. Bonfils, C. Depierreux, P. Midoux,N. T. Thuong, M. Monsigny and A. C. Roche, Nucl. Acids Res. 20,4621-4629 (1992). Similarly, oligodeoxynucleotides modified at the 3'end with biotin were combined with mannose-modified streptavidin, andwere also found to have facilitated entry into macrophages; see E.Bonfils, C. Mendes, A. C. Roche, M. Monsigny and P. Midoux, Bioconj.Chem., 3, 277-284 (1992).

Various peptides and proteins, many of which are naturally occurring,have been shown to have receptors on cell surfaces, that once they areattached thereto, allow them to become internalized by endocytosis.Materials bound to these receptors are delivered to endosomalcompartments inside the cell. Examples include insulin, vasopressin, lowdensity lipoprotein, epidermal growth factor and others. Thisinternalization has also been used to facilitate entry of DNA intocells; e.g., insulin has been conjugated to polylysine to providefacilitated DNA entry into cells possessing an insulin receptor; see B.Huckett, M. Ariatti and A. O. Hawtrey, Biochem. Pharmacol., 40, 253-263(1990).

SUMMARY OF THE INVENTION

The present invention relates to a multifunctional molecular complex forthe transfer of a nucleic acid composition to a target cell comprisingin any functional combination: 1) said nucleic acid composition; 2) oneor more cationic polyamine components bound to said nucleic acidcomposition, each comprising from three to twelve nitrogen atoms; 3) oneor more endosome membrane disruption promoting components attached to atleast one nitrogen atom of at least one of said polyamine components,through an alkyl, carboxamide, carbamate, thiocarbamate, or carbamoylbridging group, comprising a) at least one lipophilic long chain alkylgroup, b) a fusogenic peptide comprising spike glycoproteins ofenveloped animal viruses, or c) cholic acid or cholesteryl orderivatives; and optionally 4) one or more receptor specific bindingcomponents which are ligands for natural receptors of said target cell,attached through an alkyl, carboxamide, carbamate, thiocarbamate, orcarbamoyl bridging group to either a) a further nitrogen atom of atleast one of said polyamine components to which said one or moreendosome membrane disruption promoting components is attached, or b) anitrogen atom of at least one further polyamine component which does nothave attached thereto any endosome membrane disruption promotingcomponent.

The present invention further relates to a self-assembling deliverysystem for the transfer of a nucleic acid composition to a target cellcomprising the following separate components capable of being broughttogether and chemically joined into a molecular complex by simplemixing: A) said nucleic acid composition to be transferred; and B) adelivery vehicle, referred to herein as the "transfer moiety",comprising a) one or more cationic polyamine components which will bind,i.e., which are capable of being bound to said nucleic acid composition,each comprising from three to twelve nitrogen atoms; b) one or moreendosome membrane disruption promoting components attached to at leastone nitrogen atom of at least one of said polyamine components, throughan alkyl, carboxamide, carbamate, thiocarbamate, or carbamoyl bridginggroup, comprising i) at least one lipophilic long chain alkyl group, ii)a fusogenic peptide comprising spike glycoproteins of enveloped animalviruses, or iii) cholic acid or cholesteryl or derivatives; andoptionally 4) one or more receptor specific binding components which areligands for natural receptors of said target cell, attached through analkyl, carboxamide, carbamate, thiocarbamate, or carbamoyl bridginggroup to either a) a further nitrogen atom of at least one of saidpolyamine components to which said one or more endosome membranedisruption promoting components is attached, or b) a nitrogen atom of atleast one further polyamine component which does not have attachedthereto any endosome membrane disruption promoting component.

The present invention also includes the transfer moiety, described indetail immediately above, as a separate composition of matter.

The present invention also relates to a method for the transfer of anucleic acid composition to target cells on an in vitro basis. Themethod comprises the step of contacting said target cells with amultifunctional molecular complex which includes said nucleic acidcomposition, as detailed further above, thereby transferring to saidcells, a nucleic acid molecule that comprises a nucleotide sequence thateither encodes a desired peptide or protein, or serves as a template forfunctional nucleic acid molecules. The desired protein or functionalnucleic acid molecule may be any product of industrial, commercial orscientific interest, e.g., therapeutic agents including vaccines;foodstuffs and nutritional supplements; compounds of agriculturalsignificance such as herbicides and plant growth regulants,insecticides, miticides, rodenticides, and fungicides; compounds usefulin animal health such as parasiticides including nematocides; and soforth. The target cells are typically cultures of host cells comprisingmicrooganism cells such as bacteria and yeast, but may also includeplant and mammalian cells. The cell cultures are maintained inaccordance with fermentation techniques well known in the art, whichmaximize production of the desired protein or functional nucleic acidmolecule, and the fermentation products are harvested and purified byknown methods.

The present invention further relates to a method for the transfer of anucleic acid composition to the cells of an individual. The methodcomprises the step of contacting cells of said individual with amultifunctional molecular complex which includes said nucleic acidcomposition, as detailed further above, thereby administering to thecells, a nucleic acid molecule that comprises a nucleotide sequence thateither encodes a desired peptide or protein, or serves as a template forfunctional nucleic acid molecules. The nucleic acid molecule isadministered free from retroviral particles. The desired protein mayeither be a protein which functions within the individual or serves toinitiate an immune response. The nucleic acid molecule may beadministered to the cells of said individual on either an in vivo or exvivo basis, i.e., the contact with the cells of the individual may takeplace within the body of the individual in accordance with theprocedures which are most typically employed, or the contact with thecells of the individual may take place outside the body of theindividual by withdrawing cells which it is desired to treat from thebody of the individual by various suitable means, followed by contactingof said cells with said nucleic acid molecule, followed in turn byreturn of said cells to the body of said individual.

The present invention also concerns a method of immunizing an individualagainst a pathogen. The method comprises the step of contacting cells ofsaid individual with a multifunctional molecular complex which includesa nucleic acid composition, as detailed further above, therebyadministering to the cells, a nucleic acid molecule that comprises anucleotide sequence that encodes a peptide which comprises at least anepitope identical to, or substantially similar to an epitope displayedon said pathogen as antigen, and said nucleotide sequence is operativelylinked to regulatory sequences. The nucleic acid molecule is capable ofbeing expressed in the cells of the individual.

The present invention relates to methods of immunizing an individualagainst a hyperproliferative disease or an autoimmune disease. Themethods comprise the step of contacting cells of said individual with amultifunctional molecular complex which includes a nucleic acidcomposition, as detailed further above, thereby administering to cellsof the individual, a nucleic acid molecule that comprises a nucleotidesequence that encodes a peptide that comprises at least an epitopeidentical to or substantially similar to an epitope displayed on ahyperproliferative disease-associated protein or an autoimmunedisease-associated protein, respectively, and is operatively linked toregulatory sequences. The nucleic acid molecule being capable of beingexpressed in the cells.

The present invention also relates to methods of treating an individualsuffering from an autoimmune disease comprising the steps of contactingcells of said individual with a multifunctional molecular complex whichincludes a nucleic acid composition, as detailed further above, therebyadministering to cells of said individual, a nucleic acid molecule thatcomprises a nucleotide sequence which restores the activity of anabsent, defective or inhibited gene, or which encodes a protein thatproduces a therapeutic effect in the individual, and is operativelylinked to regulatory sequences; the nucleic acid molecule being capableof being expressed in said cells.

The present invention still further relates to pharmaceuticalcompositions which comprise a multifunctional molecular complex whichincludes a nucleic acid composition, as detailed further above,including pharmaceutically acceptable salt and ester forms of saidmolecular complex, together with a pharmaceutically acceptable carrier.In this regard, the present invention also relates to pharmaceuticalkits which comprise a container comprising a nucleic acid composition,and a container comprising a transfer moiety. Optionally, there isincluded in such kits excipients, carriers, preservatives and vehiclessuch as solvents.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one embodiment of the present invention there isprovided a multifunctional molecular complex for the transfer of anucleic acid composition to a target cell, which provides for a highlevel of transfection and expression of the nucleic acid molecules inthe target, i.e., host cell. This multifunctional molecular complexcomprises essentially the combination of two key elements, (I) thenucleic acid composition which it is desired to transfer to the targetcell, and (II) the transfer moiety, which complexes with the nucleicacid molecule, and comprises several components whose function is i) tolocate the desired target cell within the body of an individual by meansof a receptor specific binding component responsive to a specificreceptor on the membrane surface of said target cell; ii) to overcomethe incompatibility arising from the hydrophilic nature of the nucleicacid molecule and the lipophilic nature of the cell membrane so that theformer can pass through the latter; and iii) to prevent degradation ofthe nucleic acid molecule in a lysosome of said target cell, bydisrupting the pre-lysosome, endosome formation stage, which isaccomplished by means of an endosome membrane disrupting component whichpermits the multifunctional molecular complex to escape from an endosomeformed as a result of the target cell's process of endocytosis orpinocytosis, whereby the multifunctional molecular complex enters thetarget cell and is incorporated into said endosome.

The components of the transfer moiety are as follows: A) a cationicpolyamine component bound to said nucleic acid composition, comprisingfrom three to twelve nitrogen atoms; B) an endosome membrane disruptionpromoting component comprising at least one lipophilic long chain alkylgroup attached to a nitrogen atom of said polyamine, or a shorter alkylbridging group having a terminal carboxyl, amino, hydroxyl or sulfhydrylgroup to which there is attached a fusogenic peptide, or cholic acid orcholesteryl or derivative; and optionally C) one or more receptorspecific binding components which are ligands for natural receptors ofsaid target cell, attached to a shorter alkyl bridging group attached toa further nitrogen atom of said polyamine, through said terminal groupthereof.

The transfer moiety may be represented as one or more independentlyselected cationic polyamine components of the formula (1):

    NR(R.sup.3)-- --(CR.sup.1 R.sup.2).sub.m --N(R.sup.3)--!.sub.n --(CR.sup.1 R.sup.2).sub.m --NR(R.sup.3)                              (1)

wherein:

R, R¹ and R² are each independently selected from the group consistingof hydrogen and C₁₋₆ alkyl;

m in each occurrence is independently selected from the integers 2through 5 inclusive; and is preferably 3 or 4;

n is selected from the integers 1 through 10 inclusive; and ispreferably 1 to 6;

R³ is independently selected from the group consisting of hydrogen; C₁₋₆alkyl; and one or more endosome membrane disruption promoting componentsindependently selected from the group consisting of:

a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are each independentlydefined as above; j is an integer from 6 to :24 inclusive, preferably 8to 18, more preferably 8 to 12 inclusive; and B is optionally absent, oris a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, preferably 3 to 5, l is aninteger from 0 to 4 inclusive, preferably 0 to 2, and p is an integerfrom 1 to 3 inclusive, preferably 1; R, R¹ and R² are each independentlydefined as above; and Z is O, S, N(R), or is absent, i.e., a singlebond;

b) --B-- (R⁴)R, where R, R¹ and R² are each independently defined asabove; B cannot be absent and is a bridging group independently selectedfrom groups i) through iv) above, and additionally from the group of theformula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive, preferably 2 to 6inclusive, and more preferably 5; R, R¹, and R² are each independentlydefined as above;

X is O, S, N(R), or absent; and

R⁴ is independently selected from the group consisting of:

i) fusogenic peptides comprising spike glycoproteins of enveloped animalviruses;

ii) cholic acid derivatives of the formula (2): ##STR1## where:

represents a bond of unspecified stereochemistry;

- - - represents a single or double bond, i.e., a saturated orunsaturated portion of the ring system, provided that they cannot bothbe unsaturated at the same time, i.e., the ring system must be either Δ4or Δ5;

R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂O)_(n') H, where n' is an integer from 1 to 6 inclusive;

R⁷ is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and

R⁸ is C₁₋₆ alkyl, especially CH₃ ; including the preferred cholic acidderivatives 3α,7α, 12α-trihydroxy-5β-cholan-24-oic ester or amide; andiii) cholesteryl derivatives of the formula (3): ##STR2## where:

represents a bond of unspecified stereochemistry;

- - - represents a single or double bond, i.e., a saturated orunsaturated portion of the ring system, provided that they cannot bothbe unsaturated at the same time, i.e., the ring system must be either Δ4or Δ5;

R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O)--;

R^(7a) is C₁₋₆ alkyl, especially (CH₂)₃ CH(CH₃)₂ ; and

R^(8a) is C₁₋₆ alkyl, especially CH₃ ; including the preferredcholesteryl derivatives cholest-5-en-3'-β-carbonate, -β-carbamate, or-β-methylenecarboxamide;

PROVIDED THAT R³ is one or more endosome membrane disruption promotingcomponents attached to at least one nitrogen atom of at least one ofsaid cationic polyamine components; and

OPTIONALLY, R³ may be one or more groups defined below, attached eitherto a further nitrogen atom of at least one of said cationic polyaminecomponents to which said one or more endosome membrane disruptionpromoting components is attached, or to a nitrogen atom of at least onefurther polyamine component which does not have attached thereto anyendosome membrane disruption promoting component:

c) --B--(R⁵)R, where B cannot be absent, and is a bridging groupindependently selected from groups i) through v) inclusive; R isindependently defined as above; and

R⁵ is a receptor specific binding component independently selected fromthe group consisting of:

i) D-biotin;

ii)β-3'-propionyl galactosyl-β1-4-thioglucoside;

iii) N² N⁶ -bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine;

iv) N²,N⁶ -bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine;

v) 5-methyitetrahydrofolate;

vi) folic acid;

vii) folinic acid;

viii) α-3'-propionyl thiomannoside; and

ix) α-3'-propionyl thiomannoside-6-phosphate.

The Nucleic Acid Composition

The two basic components of the multifunctional molecular complex of thepresent invention are the nucleic acid composition and the transfermoiety. By "nucleic acid composition" is meant any one or more of thegroup of compounds in which one or more molecules of phosphoric acid arecombined with carbohydrate, i.e., pentose or hexose, molecules, whichare in turn combined with bases derived from purine, e.g., adenine, andfrom pyrimidine, e.g., thymine. Particular naturally occurring nucleicacid molecules include genomic deoxyribonucleic acid (DNA) and genomicribonucleic acid (RNA), as well as the several different forms of thelatter, e.g., messenger RNA (mRNA), transfer RNA (tRNA), and ribosomalRNA (rRNA). Also included are the different DNA's which arecomplementary (cDNA) to the different RNA's. Synthesized DNA or a hybridthereof with naturally occurring DNA, is contemplated.

The nucleic acid compositions used in the present invention may beeither single-stranded or double-stranded, may be linear or circular,e.g., a plasmid, and are either oligo- or polynucleotides. They maycomprise as few as 15 bases or base pairs, or may include as many as 20thousand bases or base pairs (20 kb). Since the transfer moiety isemployed on a pro rata basis when added to the nucleic acid composition,practical considerations of physical transport will largely govern theupper limit on the size of nucleic acid compositions which can beutilized.

In addition to these naturally occurring materials, the nucleic acidcompositions used in the present invention can also include syntheticcompositions, i.e., nucleic acid analogs. These have been found to beparticularly useful in antisense methodology, which is the complementaryhybridization of relatively short oligonucleotides to single-strandedRNA or single-stranded DNA, such that the normal, essential functions ofthese intracellular nucleic acids are disrupted. See, e.g., Cohen,Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press,Inc., Boca Raton, Fla. (1989).

The size, nature and specific sequence of the nucleic acid compositionto be transferred to the target cell can be optimized for the particularapplication for which it is intended, and such optimization is wellwithin the skill of the artisan in this field. However, the nature ofthe target cells within the individual into which it is desired totransfer a nucleic acid composition, may have a significant bearing onthe choice of the particular multifunctional molecular complex of thepresent invention. For example, where it is desired to transfer nucleicacid molecules to target cells by injecting them intramuscularly toevoke an immune response, it will be found that this transfer can beeffected by use of a multifunctional molecular complex of the presentinvention, as defined above, comprising a cationic polyamine to which isattached, as the endosome membrane disruption promoting component, alipophilic long chain alkyl group as defined above. Where the targetcells are hepatocytes, for example, transfer of the desired nucleic acidcomposition is readily effected by use of the multifunctional molecularcomplex of the present invention wherein there is attached to thecationic polyamine a receptor specific binding component which willpermit discrimination among body cells, comprising, e.g., N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine, or N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside) lysyl-N⁶ -(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine.

The nucleic acid composition to be transferred to a target cell inaccordance with the present invention must have an appropriate openreading frame and promoter to express a protein, as well as any otherregulatory sequences which may be appropriate to expression. Nucleicacid compositions to be delivered by means of the methods of the presentinvention can be designed and constructed so as to be appropriate forthe particular application desired, all of which is well within theordinary skill of the artisan in this field.

The nucleic acid molecules which are delivered to cells using themultifunctional molecular complex and methods of the present inventionmay serve as: 1) genetic templates for proteins that function asprophylactic and/or therapeutic immunizing agents; 2) replacement copiesof defective, missing or non-functioning genes; 3) genetic templates fortherapeutic proteins; 4) genetic templates for antisense molecules andas antisense molecules per se; or 5) genetic templates for ribozymes.

In the case of nucleic acid molecules which encode proteins, the nucleicacid molecules preferably comprise the necessary regulatory sequencesfor transcription and translation in the target cells of the individualanimal to which they are delivered.

In the case of nucleic acid molecules which serve as templates forantisense molecules and ribozymes, such nucleic acid molecules arepreferably linked to regulatory elements necessary for production ofsufficient copies of the antisense and ribozyme molecules encodedthereby respectively. The nucleic acid molecules are free fromretroviral particles and are preferably provided as DNA in the form ofplasmids.

The Transfer Moiety

The core, or backbone of the transfer moiety is-the cationic polyamine,containing between 3 and 12 amines. There may be more than one of thesecationic polyamine components, whose function is to overcome theincompatibility arising from the hydrophilic nature of the nucleic acidmolecule and the lipophilic nature of the cell membrane, although thisby itself will not permit the former to pass through the latter. Thecationic groups of the polyamine bind to the anionic groups of thenucleic acid through ionic bonding, thus neutralizing those charges andalso serving as a point of attachment for the complex. One μg of DNAcontains 3.1 nanomoles of phosphate anionic charges, assuming a meanmolecular weight of 325 for a nucleotide sodium salt. The transfermoiety of the present: invention will not become effective in achievingtransfer of the nucleic acid composition until the anionic charges ofsaid nucleic acid are substantially neutralized by the cationic chargesof the polyamine component of the transfer moiety.

It will be appreciated that in one embodiment of the present invention,a single cationic polyamine can be employed which, conceptually,balances the anionic charges of the nucleic acid in a more or lessstoichiometric fashion, although it will be understood that, as apractical matter, it will be necessary to employ amounts of cationicpolyamine which are significantly in excess of the stoichimetric amount,because of the presence of competing binding sites in target and othercells, whose existence is well known to the artisan and whichcompetitively prevent or otherwise interfere with the binding of thepolyamine to the nucleic acid as desired. It is also contemplated thatmore than one such cationic polyamine can be employed, in which caseeach polyamine chain or piece is smaller than the corresponding nucleicacid to which it will become bound. It will be understood, however, thatthe total size or length of these individual cationic polyaminecomponents should together be substantially the same size or length asthe nucleic acid component, in order for neutralization of the anioniccharges of the nucleic acid to take place. Again, it will be understoodthat for practical reasons, a significant excess of cationic polyaminecomponents, over the amount of nucleic acid component present, will benecessary. Using more than one cationic polyamine component permitsflexibility with respect to the types of groups that are attachedthereto. For example, one cationic polyamine component may carry aparticular endosome membrane disruption promoting component, whileanother cationic polyamine component caries a receptor specific bindingcomponent, or perhaps a different endosome membrane disruption promotingcomponent. The total number of such cationic polyamine components isvariable, and will depend not only on the size or length of the nucleicacid component, but on the number and type of groups attached thereto aswell.

Transfer efficiency, i.e., transfection, does not become optimum untilthe multifunctional molecular complex, the combination of the transfermoiety and the nucleic acid, bears a strong positive charge. Thus, theamount of transfer moiety must be selected with this in mind, and theactual amount chosen be depend on the charge density thereof, which canbe calculated by means well known in the art.

The triamine, tetraamine, pentaamine and higher polyamine components ofthe transfer moiety must be cationic in order to be functional, asexplained above. This can be accomplished by the simple expedient ofmaking an acid addition salt, e.g., the hydrochloride salt, whereammonium chloride units are formed. It may also be the case that thecationic form of the polyamine is formed under conditions of physiologicpH, in which case it is not necessary to form the cation directly. Thus,the term "cationic polyamine" is intended to include both of thesepossibilities.

It is contemplated that the number of amine groups that it is desired tohave present in the polyamine will depend to some extent on the mode ofadministration of the multifunctional molecular complex that is used.For example, it is contemplated that for intramuscular administration,it is preferred to have from 3 to 5 amine groups in the polyamine;whereas, for systemic injection, e.g., intravenous injection, it ispreferred to have from 5 to 8 amine groups in the polyamine. For invitro applications generally, it is preferred to have from 5 to 8 aminegroups in the polyamine.

The next component of the transfer moiety is the endosome membranedisruption promoting component, which is required to be present. Thiscan either comprise one or more lipophilic long chain alkyl groupsattached through one or more of the nitrogen atoms of said polyamine, orcan comprise a bridging group "B", e.g., a shorter alkyl linking moiety,optionally with a terminal amino, hydroxyl or sulfhydryl group, throughwhich there is attached a fusogenic peptide, or cholic acid orcholesteryl or derivative compound.

The lipophilic long chain alkyl group is defined by the formula:--B--(CR¹ R²)_(j) --C(R)₃, where B is a bridging group as defined; R, R¹and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; and j is an integer from 6 to 24 inclusive,preferably 8 to 18, more preferably 8 to 12 inclusive.

The group "--B--" may be absent, i.e., a single bond, where R³ is theendosome membrane disruption promoting component comprising a lipophiliclong chain alkyl group as defined under "a)" above. The group "--B--"may also be a bridging element which is a member independently selectedfrom the group consisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--N(R)--;            i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--O--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--(CH.sub.2).sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--;                                            iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --C(═O)}.sub.p --N(R)--;iv)

or

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where the various substituents are as defined above.

Where the endosome membrane disruption promoting component, rather thanbeing a lipophilic long chain alkyl group, is instead a fusogenicpeptide or a cholic acid or cholesteryl or derivative compound, thebridging group "B" is required to be present, and will be a memberindependently selected from the group i) through v) above. Thisselection will be dependent upon the required or desired type ofchemical linkage to be present. For example, members i) and iv) arecarboxamide linkages, whereas members ii) and iii) are carbamate,thiocarbamate, or carbamoyl linkages, depending upon whether "Z" is O, Sor absent, respectively. For member v), the linkages will be oxy, thio,amino, or alkylene, depending upon whether "X" is O, S, N(R), or absent,respectively. The endosome membrane disruption promoting component, onthe other hand, may have a carbonyl, amino, or some other terminalgroup, which can determine the choice of bridging member to be used. Allsuch choices, however, are well within the skill of the artisan in thisfield.

Most simply, the bridging group can be an alkylene linking moiety usedprimarily for steric considerations. However, the other bridging groupsmay also be desirable for imparting various physical and chemical, aswell as configurational properties to the multifunctional molecularcomplex of the present invention. The polyethylene glycol group can beespecially useful in this regard.

The term "C₁₋₆ alkyl", as used above, and throughout the description ofthe present invention, refers to straight and branched chain alkylgroups including, but not limited to methyl, ethyl, propyl, isopropyl,n-butyl, iso-butyl, sec-butyl, t-butyl, and n-pentyl.

In the formula for the lipophilic long chain alkyl group above, it ispreferred that R, R¹ and R² are all hydrogen, and, as indicated that jbe an integer from 8 to 18 inclusive. There must be at least one ofthese lipophilic long chain alkyl groups present, but preferably thereare no more than three such groups present. It is preferred to have onlyone such group. Thus, examples of preferred transfer moieties of thepresent invention, where the endosome membrane disruption promotingcomponent is a lipophilic long chain alkyl group as described above, areN-octylspermidine, N⁴ -dodecylspermidine, N⁴ -octadecylspermidine, N⁴-octylspermine, N⁴ -dodecylspermine, and N⁴ -octadecylspermine.

The endosome membrane disruption promoting component can also comprise ashorter alkyl bridging group, optionally having a terminal amino,hydroxyl or sulfhydryl group through which there is attached a fusogenicpeptide, or cholesterol or derivative compound. Such a component may berepresented by the formula --B-- (R⁴)R, where the B group is --(CR¹R²)_(j') --X--, where R, R¹ and R² are each independently selected fromthe group consisting of hydrogen and C₁₋₆ alkyl; j' is an integer from 1to 6 inclusive, preferably 2 to 4 inclusive; X is O, S, N(R), or absent.

It is preferred that R, R¹ and R² each be hydrogen, and as indicated,that j' be 2 to 4, while X is defined as N. Thus, the shorter alkylbridging group will preferably be ethyl, n-propyl, or n-butyl, and willhave a terminal amino group to which is attached the fusogenic peptide,or cholic acid or cholesteryl or derivative compound, which comprisesthe endosome membrane disruption promoting component.

Alternatively, other of the members of the "B" bridging group can bechosen. For example, member i) provides an alkyl bridging moiety with acarboxamide linkage, the most simple representative of which would bethe group --(CH₂)--C(═O)--NH--. Member ii) provides an alkylene.bridging moiety with a carbamate type of linkage; and the most simplerepresentative of this member would be the group --(CH₂)--NH--C(═O)--,which is a carbamoyl type of linkage. A carbamate linkage would berepresented by the group --(CH₂)--NH--C(═O)--O--, while a simple variantof this group would provide a thiocarbamate linkage:--(CH₂)--NH--C(═O)--S--. Members iii) and iv) provide the same terminallinkage variants, while adding to the alkyl bridging moiety apolyethylene glycol bridging moiety of variable size, i.e., number ofrepeating ethylene oxide monomer units, depending upon the definitionsof "l" and "p".

The fusogenic peptide which functions as an endosome membrane disruptionpromoting component, comprises the spike glycoproteins of envelopedanimal viruses known in the art. Membrane fusion, whether planar orannular, comprises the stages of initial approach, coalescence, andseparation. Fusion reactions are rapid, highly specific, and non-leaky.The membrane proteins of enveloped animal viruses comprise glycoproteinswhich span the bilayer of the virus membrane and have the bulk of theirmass externally, and non-spanning, nonglycosylated proteins associatedwith the inner bilayer surface. The glycoproteins form radialprojections on the surface of the virus membrane, and these spikeglycoproteins play a key role in virus entry into host cells. Spikeglycoproteins are among the best-characterized virus membrane proteins.In cell entry the spike glycoproteins are responsible for attachment ofthe virus particle to the cell surface, and for penetration of thenucleocapsid into the cytosol, where, after endocytosis of the virusparticle, the spike glycoproteins play a role in fusion with thelimiting membrane of the endosome, whereby the nucleocapsid reenters thecytosol. In some enveloped animal viruses, the spike glycoproteins takeon a specialized character, e.g., in orthomyxoviruses, where one is aneuraminidase and another is a haemagglutinin. All of these fusogenicpeptides, in terms of their amino acid sequences, gross morphology, rolein the overall process of fusion, and requirements for activity, havebeen the subject of long term study and have been disclosed in detail inthe technical literature. See, e.g., J. White, M. Kielian and A.Helenius, Quarterly Reviews of Biophysics, 16, 151-195 (1983), which isincorporated herein by reference in its entirety.

The cholic acid and derivatives which function as endosome membranedisruption promoting components, comprise compounds of the formula (2):##STR3## where:

represents a bond of unspecified stereochemistry;

- - - represents a single or double bond, i.e., a saturated orunsaturated portion of the ring system, provided that they cannot bothbe unsaturated at the same time, i.e., the ring system must be either Δ4or Δ5;

R⁶ is H, OH, CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂O)_(n') H, where n' is an integer from 1 to 6 inclusive;

R⁷ is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and

R⁸ is C₁₋₆ alkyl, especially CH₃. It is preferred that the cholic acidand derivative compounds comprise one or more members selected from thegroup consisting of 3α,7α,12α-trihydroxy-5α-cholan-24-oic ester andamide.

The cholesteryl and derivatives which function as endosome membranedisruption promoting components, comprise compounds of the formula (3):##STR4## where:

represents a bond of unspecified stereochemistry;

- - - represents a single or double bond, i.e., a saturated orunsaturated portion of the ring system, provided that they cannot bothbe unsaturated at the same time, i.e., the ring system must be either Δ4or Δ5;

R^(6a) is a radical that forms the point of attachment of the cholicacid derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O)--;

R^(7a) is C₁₋₆ alkyl, especially (CH₂)₃ CH(CH₃)2;and

R^(8a) is C₁₋₆ alkyl, especially CH₃. It is preferred that. thecholesteryl and derivative compounds comprise one or more membersselected from the group consisting of cholest-5-en-3'-β-carbonate,-β-carbamate, and -βmethylene-carboxamide.

An optional embodiment of the present invention is to provide for areceptor specific binding component which helps effect transfer ofnucleic acid compositions to target cells, especially eukaryotic cells,by taking advantage of the natural receptor-mediated endocytosispathways which exist in those cells. The receptor specific bindingcomponent is thus a ligand for the natural receptor, and can thus assistin binding of the multifunctional molecular complex to the target cell.Endocytosis or pinocytosis will then take place whereby the entirecomplex is transferred into the target cell, enclosed in an endosome.

The receptor specific binding component serves the important function ofallowing the multifunctional molecular complex of the present inventionto be targeted to specific cell populations, e.g., hepatocytes. Thebinding component facilitates location of the desired target cellswithin the body of the animal to which the complex is beingadministered, with subsequent attachment of the complex to the targetcells.

Where the receptor specific binding component is employed, there willalso be present on the multifunctional molecular complex an endosomemembrane disruption promoting component, as defined further above.Accordingly, once the binding component has located the desired targetcell within the individual, and attached the complex to said cell bybinding to said receptor, the complex will be transferred into said cellby endocytosis, whereupon it will be enclosed within an endosome. Atthis point, the endosome membrane disruption promoting component assumesits important role by disrupting said membrane, allowing escape of thecomplex into the cytoplasm of said cell.

During the normal course of events in the target cell, endosomeformation is a prelude to targeting of any foreign protein to lysosomeswhere degradation of the foreign protein by hydrolytic enzymes will takeplace. Consequently, accumulation in lysosomal compartments can be amajor obstacle to the effectiveness of nucleic acid delivery systems.The multifunctional molecular complex of the present invention wouldsuffer the same fate, were it not for the presence of the endosomemembrane disruption promoting component. This component permits thecomplex to escape from the endosome, whereupon it can migrate into thenucleus of the target cell, and release the nucleic acid composition,whose genetic information can then be transcribed within said nucleus.Although the precise mechanisms which make up these steps and pathwaysare not well understood, expression of the nucleic acid moleculecontained in the multifunctional molecular complex does take place, asis demonstrated in the working examples further below.

The receptor specific binding component may be represented by theformula: --B--(R⁵)R, where R, R¹ and R² are each independently hydrogenor C₁₋₆ alkyl; B may be, inter alia, --(CR¹ R²)_(j') --X--, where R¹ andR² are as defined above, X is N(R), and j' is an integer from 1 to 6inclusive, preferably 2 to 4 inclusive; and R⁵ is a receptor specificbinding component independently selected from the group consisting of:

i) D-biotin;

ii) β-3'-propionyl galactosyl-β1-4-thioglucoside;

iii) N²,N⁶ -bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine;

iv) N²,N⁶ -bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine;

v) 5-methyltetrahydrofolate;

vi) folic acid;

vii) folinic acid;

viii) α-3'-propionyl thiomannoside; and

ix) α-3'-propionyl thiomannoside-6-phosphate.

It is preferred that R, R¹ and R² each be hydrogen, and as indicated,that j' be 2 to 6. Thus, the shorter alkyl bridging group willpreferably be ethyl, n-propyl, or n-butyl, and the receptor specificbinding component is attached to the terminal amino group.

Since the endosome membrane disruption promoting component must also bepresent, examples of preferred transfer moieties of the presentinvention, where the receptor specific binding component is a galactosylgroup as described above, are

N² -octyl-N⁴ -(5-(β-3'-propionyl galactosyl-β1"-4'-thioglucoside) amino)pentylspermidine;

N² -dodecyl-N⁴ -(5-(β-3'-propionylgalactosyl-β1"-4'-thioglucoside)amino)pentylspermidine;

N⁶ -octadecyl-N⁴ -(5-(β-3'-propionylgalactosyl-β1"-4'-thioglucoside)amino)pentyl-spermidine;

N⁶ -octyl-N⁴ -(5- N^(2'),N^(6') -bis(β1-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N^(6") -(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!amino)pentyl-spermine;

N² -dodecyl-N⁴ -(5- N^(2'),N^(6') -bis(β1-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N^(6') -(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!amino)pentyl-spermine; and

N² -octadecyl-N⁴ -(5- N^(2'),N^(6') -bis(β1-3'-propionylgalactosyl-β1-4-thioglucoside) lysyl-N^(6') -(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!amino)pentyl-spermine.

Accordingly, there has been described in detail above themultifunctional molecular complex of the present invention for transferof nucleic acid compositions to a target cell. This complex includes thetransfer moiety as a separate, distinct embodiment of the presentinvention. Further aspects of the present invention relate to themethods of using the multifunctional molecular complex.

Thus, in accordance with the present invention there is provided amethod for the transfer of a nucleic acid composition to target cells onan in vitro basis. In this method target cells are contacted with amultifunctional molecular complex which includes said nucleic acidcomposition. In one embodiment, the target cells have been isolated froman individual, and all of the cells are thus of the same type, and it isnot necessary, therefore, for the complex to include a receptor specificbinding component. An especially preferred embodiment is one in which amicroorganism culture is maintained under fermentation conditions, and aprotein product is expressed by the microorganism as a result of thetransfer thereto of nucleic acid compositions using the multifunctionalmolecular complex of the present invention. The protein product isisolated and purified. Here again, a single type of target cell isinvolved, so that it is not necessary that a receptor specific bindingcomponent be present.

This method provides for transfer to target cells of a nucleic acidmolecule that comprises a nucleotide sequence that either encodes adesired peptide or protein, or serves as a template for functionalnucleic acid molecules. The desired protein or functional nucleic acidmolecule may be any product of industrial, commercial or scientificinterest, e.g., therapeutic agents including vaccines; foodstuffs andnutritional supplements; compounds of agricultural significance such asherbicides and plant growth regulants, insecticides, miticides,rodenticides, and fungicides; compounds useful in animal health such asparasiticides including nematocides; and so forth. The target cells aretypically cultures of host cells comprising microoganism cells such asbacteria and yeast, but may also include plant and mammalian cells. Thecell cultures are maintained in accordance with fermentation techniqueswell known in the art, which maximize production of the desired proteinor functional nucleic acid molecule, and the fermentation products areharvested and purified by known methods.

The present invention further relates to a method for the transfer of anucleic acid composition to the cells of an individual in an in vivomanner. The method comprises the step of contacting cells of saidindividual with a multifunctional molecular complex of the presentinvention, which includes said nucleic acid composition. Here again, thenucleic acid molecule comprises a nucleotide sequence that eitherencodes a desired peptide or protein, or serves as a template forfunctional nucleic acid molecules. The nucleic acid molecule isadministered free from retroviral particles. The desired protein mayeither be a protein which functions within the individual or serves toinitiate an immune response.

The nucleic acid molecule may be administered to the cells of saidindividual on either an in vivo or ex vivo basis, i.e., the contact withthe cells of the individual may take place within the body of theindividual in accordance with the procedures which are most typicallyemployed, or the contact with the cells of the individual may take placeoutside the body of the individual by withdrawing cells which it isdesired to treat from the body of the individual by various suitablemeans, followed by contacting of said cells with said nucleic acidmolecule, followed in turn by return of said cells to the body of saidindividual.

The method of transferring a nucleic acid composition to the cells of anindividual provided by the present is invention, includes particularly amethod of immunizing an individual against a pathogen. In this method,the nucleic acid composition administered to said cells, comprises anucleotide sequence that encodes a peptide which comprises at least anepitope identical to, or substantially similar to an epitope displayedon said pathogen as antigen, and said nucleotide sequence is operativelylinked to regulatory sequences. The nucleic acid molecule must, ofcourse, be capable of being expressed in the cells of the individual.

The method of transferring a nucleic acid composition to the cells of anindividual provided by the present invention, further includes methodsof immunizing an individual against a hyperproliferative disease or anautoimmune disease. In such methods, the nucleic acid composition whichis administered to the cells of the individual comprises a nucleotidesequence that encodes a peptide that comprises at least an epitopeidentical to or substantially similar to an epitope displayed on ahyperproliferative disease-associated protein or an autoimmunedisease-associated protein, respectively, and is operatively linked toregulatory sequences. Here again, the nucleic acid molecule must becapable of being expressed in the cells of the individual.

In accordance with the present invention there is also provided methodsof treating an individual suffering from an autoimmune disease, in whichthe cells of said individual are contacted with a multifunctionalmolecular complex including a nucleic acid composition, therebyadministering a nucleic acid molecule that comprises a nucleotidesequence which restores the activity of an absent, defective orinhibited gene, or which encodes a protein that produces a therapeuticeffect in said individual, and is operatively linked to regulatorysequences; the nucleic acid molecule being capable of being expressed insaid cells.

In order to carry out the methods described above, the present inventionprovides pharmaceutical compositions which comprise the multifunctionalmolecular complex including a nucleic acid composition, as well aspharmaceutically acceptable salt and ester forms of said molecularcomplex, together with a pharmaceutically acceptable carrier. Alsoincluded are kits which comprise a container comprising a nucleic acidcomposition, and a container comprising a transfer moiety, as describedherein. Optionally, there is included in such kits excipients, carriers,preservatives and vehicles such as solvents.

Accordingly the present invention provides compositions and methodswhich prophylactically and/or therapeutically immunize an individualagainst a pathogen or abnormal, disease-related cell. The geneticmaterial encodes a peptide or protein that shares at least an epitopewith an immunogenic protein found on the pathogen or cells to betargeted. The genetic material is expressed by the individual's cellsand serves as an immunogenic target against which an immune response iselicited. The resulting immune response is broad based: in addition to ahumoral immune response, both arms of the cellular immune response areelicited. The methods of the present invention are useful for conferringprophylactic and therapeutic immunity. Thus, a method of immunizingincludes both methods of protecting an individual from pathogenchallenge, or occurrence or proliferation of specific cells, as well asmethods of treating an individual suffering from pathogen infection,hyperproliferative disease or autoimmune disease. Thus, the presentinvention is useful to elicit broad immune responses against a targetprotein, i.e. proteins specifically associated with pathogens or theindividual's own "abnormal" cells.

The present invention is also useful in combating hyperproliferativediseases and disorders such as cancer, by eliciting an immune responseagainst a target protein that is specifically associated with thehyperproliferative cells. The present invention is further useful incombating autoimmune diseases and disorders by eliciting an immuneresponse against a target protein that is specifically associated withcells involved in the autoimmune condition.

Other aspects of the present invention relate to gene therapy. Thisinvolves compositions and methods for introducing nucleic acid moleculesinto the cells of an individual which are exogenous copies of geneswhich either correspond to defective, missing, non-functioning orpartially functioning genes in the individual, or which encodetherapeutic proteins, i.e., proteins whose presence in the individualwill eliminate a deficiency in the individual and/or whose presence willprovide a therapeutic effect on the individual. There is thus provided ameans of delivering such a protein which is a suitable, and evenpreferred alternative to direct administration of the protein to theindividual.

As used herein the term "desired protein" is intended to refer topeptides and proteins encoded by gene constructs used in the presentinvention, which either act as target proteins for an immune response,or as a therapeutic or compensating protein in gene therapy regimens.

Using the methods and compositions of the present invention, DNA or RNAthat encodes a desired protein is introduced into the cells of anindividual where it is expressed, thus producing the desired protein.The nucleic acid composition, e.g., DNA or RNA encoding the desiredprotein is linked to regulatory elements necessary for expression in thecells of the individual. Regulatory elements for DNA expression includea promoter and a polyadenylation signal. In addition, other elements,such as a Kozak region, may also be included in the nucleic acid.composition.

As used herein, the term "nucleic acid composition" refers to the DNA orRNA, or other nucleic acid molecule that comprises a nucleotide sequencewhich encodes the desired protein, and which includes initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the individual to which the construct is administered.

As used herein, the term "expressible form" refers to gene constructswhich contain the necessary regulatory elements operably linked to acoding sequence that encodes a target protein, such that when present inthe cell of the individual, the coding sequence will be expressed.

As used herein, the term "genetic vaccines" refers to a pharmaceuticalpreparation that comprises a nucleic acid composition that comprises anucleotide sequence that encodes a target protein, includingpharmaceutical preparations useful to invoke a therapeutic immuneresponse.

As used herein, the term "genetic therapeutic" refers to apharmaceutical preparation that comprises a nucleic acid compositionthat comprises a nucleotide sequence that encodes a therapeutic orcompensating protein. Alternatively, a genetic therapeutic may encodeantisense sequences which inhibit undesired gene expression. Further,genetic therapeutics may encode ribozymes.

As used herein, the term "target protein" refers to a protein againstwhich an immune response can be elicited. The target protein is animmunogenic protein which shares at least an epitope with a protein fromthe pathogen or undesirable cell-type, such as a cancer cell or a cellinvolved in autoimmune disease, against which immunization is required.The immune response directed against the target protein will protect theindividual against, and treat the individual for, the specific infectionor disease with which the target protein is associated.

As used herein, the term "sharing an epitope" refers to proteins whichcomprise at least one epitope that is identical to or substantiallysimilar to an epitope of another protein. And, the term "substantiallysimilar epitope" is meant to refer to an epitope that has a structurewhich is not identical to an epitope of a protein, but nonethelessinvokes a cellular or humoral immune response which cross reacts to thatprotein.

As used herein, the term "therapeutic protein" is meant to refer toproteins whose presence confers a therapeutic benefit to the individual.

As used herein, the term "compensating protein" is meant to refer toproteins whose presence compensates for the absence of a fullyfunctioning endogenously produced protein, due to an absent, defective,non-functioning or partially functioning endogenous gene.

When taken up by a target cell, a nucleic acid composition used in thepresent invention, which includes the nucleotide sequence encoding thedesired protein operably linked to regulatory elements, may remainpresent in the cell as a functioning extrachromosomal molecule, or itmay integrate into the cell's chromosomal DNA. DNA may be introducedinto cells where it remains as separate genetic material in the form ofa plasmid. Alternatively, linear DNA which can integrate into thechromosome may be introduced into the target cell. When introducing DNAinto the cell, reagents which promote DNA integration into chromosomesmay be added. DNA sequences which are useful to promote integration mayalso be included in the DNA molecule. Alternatively, RNA may beadministered to the cell. It is also contemplated to provide the nucleicacid. composition as a linear minichromosome including a centromere,telomeres and an origin of replication. As used herein, the terms "DNAconstruct", "nucleic acid composition" and "nucleotide sequence" aremeant to refer to both DNA and RNA molecules.

The regulatory elements necessary for gene expression of a DNA moleculeinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression. It is necessary that these elements be operably linkedto the sequence that encodes the desired proteins and that theregulatory elements are operable in the individual to whom they areadministered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements be functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence. Promoters andpolyadenylation signals used must also be functional within the cells ofthe individual.

Examples of promoters useful with the nucleic acid compositions used inthe present invention, especially in the production of a genetic vaccinefor humans, include but are not limited to, promoters from Simian Virus40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, HumanImmunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR)promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMVimmediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus(RSV), as well as promoters from human genes such as human Actin, humanMyosin, human Hemoglobin, human muscle creatine and humanmetalothionein.

Examples of polyadenylation signals useful with the nucleic acidcompositions used in the present invention, especially in the productionof a genetic vaccine for humans, include but are not limited to, SV40polyadenylation signals and LTR polyadenylation signals. In particular,the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen,San Diego Calif.), referred to as the SV40 polyadenylation signal, maybe used.

In addition to the regulatory elements required for nucleic acidmolecule expression, other elements may also be included in the DNAmolecule. Such additional elements include enhancers. The enhancer maybe selected from the group including but not limited to: human Actin,human Myosin, human Hemoglobin, human muscle creatine, and viralenhancers such as those from CMV, RSV and EBV.

Nucleic acid compositions can be provided with mammalian origin ofreplication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Plasmids pCEP4 andpREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region, whichproduces high copy episomal replication without integration. In aspectsof the invention relating to gene therapy, constructs with origins ofreplication including the necessary antigen for activation arepreferred.

In-other embodiments of the present invention relating to immunizationapplications, the nucleic acid composition contains nucleotide sequencesthat encode a target protein and further include genes for proteinswhich enhance the immune response against such target proteins. Examplesof such genes are those which encode cytokines and lymphokines such asa-interferon, γ-interferon, platelet derived growth factor (PDGF),GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4,IL-6, IL-8, IL-10 and IL-12. In some embodiments, it will be preferredthat the gene for GM-CSF be included in nucleic acid compositions usedin immunizing compositions.

An additional element may be added to the nucleic acid composition whichserves as a target for cell destruction, if it is desirable to eliminatethe cells receiving the nucleic acid composition for any reason. Aherpes thymidine kinase (tk) gene in an expressible form can be includedin the nucleic acid composition. The drug gangcyclovir can then beadministered to the individual and that drug will cause the selectivekilling of any cell producing tk, thus providing the means for theselective destruction of cells containing the nucleic acid composition.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in cells into whichthe construct is transferred. Moreover, codons may be selected which aremost efficiently transcribed in the target cell. One having ordinaryskill in the art can readily produce DNA constructs which are functionalin the target cells.

Nucleic acid compositions can be tested for expression levels in vitroby using tissue culture of cells of the same type as those to betreated. For example, if the genetic vaccine is to be administered tohuman muscle cells, muscles cells grown in culture, such as solid muscletumors cells of rhabdomyosarcoma, may be used as an in vitro model tomeasure expression level.

The nucleic acid compositions used in the present invention are notincorporated within retroviral particles. The nucleic acid compositionsare taken up by the cell without retroviral particle-mediated insertion,such as that which occurs when retrovirus particles with retroviral RNA,infects a cell. As used herein, the term "free from retroviralparticles" is meant to refer to nucleic acid compositions that are notincorporated within retroviral particles. As used herein, "dissociatedfrom an infectious agent" is meant to refer to genetic material which isnot part of a viral, bacterial or eukaryotic vector, either active,inactivated, living or dead, that is capable of infecting a cell.

In some embodiments, the nucleic acid compositions constitute less thana complete, replicatable viral genome such that upon introduction intothe cell, the nucleic acid composition possesses insufficient geneticinformation to direct production of infectious viral particles. As usedherein, the term "incomplete viral genome" is meant to refer to anucleic acid composition which contains less than a complete genome suchthat incorporation of such a nucleic acid composition into a cell doesnot constitute introduction of sufficient genetic information for theproduction of infectious virus.

In some embodiments, an attenuated viral vaccine may be delivered as anucleic acid composition which contains enough genetic material to allowfor production of viral particles. Delivery of the attenuated vaccine asa nucleic acid composition allows production of large quantities ofsafe, pure, and active immunizing product.

The present invention may be used to immunize an individual against allpathogens such as viruses, prokaryotic and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated, such as viruses, and prokaryotes such as Gonorrhoea,Listeria and Shigella. In addition, the present invention is also usefulfor immunizing an individual against protozoan pathogens, including anystage in their life cycle in which they are intracellular pathogens. Asused herein, the term "intracellular pathogen" is meant to refer to avirus or pathogenic organism that, during at least part of itsreproductive or life cycle, exists within a host cell and thereinproduces or causes to be produced, pathogenic proteins. Table 1 providesa listing of some of the viral families and genera for which vaccinesaccording to the present invention can be made. DNA constructs thatcomprise DNA sequences which encode the peptides that comprise at leastan epitope identical or substantially similar to an epitope displayed ona pathogen antigen, such as those antigens listed in said table, areuseful in vaccines.

Moreover, the present invention is also useful to immunize an individualagainst other pathogens including prokaryotic and eukaryotic protozoanpathogens as well as multicellular parasites such as those listed inTable 2.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material which encodes immunogenic proteins againstwhich a protective immune response can be mounted, must be included inthe nucleic acid composition. Whether the pathogen infectsintracellularly, for which the present invention is particularly useful,or extracellularly, it is unlikely that all pathogen antigens willelicit a protective response. Because DNA and RNA are both relativelysmall and can be produced relatively easily, the present inventionprovides the additional advantage of allowing for vaccination withmultiple pathogen antigens. The nucleic acid composition used in thegenetic vaccine can include genetic material which encodes many pathogenantigens. For example, several viral genes may be included in a singleconstruct, thereby providing multiple targets. In addition, multipleinoculants which can be delivered to different cells in an individualcan be prepared to collectively include, in some cases, a complete or,more preferably, an incomplete, e.g., nearly complete set of genes inthe vaccine. For example, a complete set of viral genes may beadministered using two constructs which each contain a different half ofthe genome which are administered at different sites. Thus, an immuneresponse may be invoked against each antigen without the risk of aninfectious virus being assembled. This allows for the introduction ofmore than a single antigen target and can eliminate the requirement thatprotective antigens be identified.

The ease of handling and inexpensive nature of DNA and RNA further allowfor more efficient means of screening for protective antigens. Genes canbe sorted and systematically tested much more easily than proteins. Oncethe pathogenic agents and organism for which a protective vaccine willbe sought is selected, an immunogenic protein is then identified. Tables1 and 2 include lists of some of the pathogenic agents and organisms forwhich genetic vaccines can be prepared to protect an individual frominfection by them. The methods of immunizing an individual against apathogen can be directed particularly against HIV, HTLV or HBV.

In accordance with the present invention there is also provided a methodof conferring a broad based protective immune response againsthyperproliferating cells that are characteristic of hyperproliferativediseases, as well as a method of treating individuals suffering fromhyperproliferative diseases. As used herein, the term"hyperproliferative diseases" is meant to refer to those diseases anddisorders characterized by hyperproliferation of cells. Examples ofhyperproliferative diseases include all forms of cancer and psoriasis.

It has been discovered that introduction of a nucleic acid compositionthat includes a nucleotide sequence which encodes an immunogenic"hyperproliferating cell"-associated protein into the cells of anindividual, results in the production of those proteins in thevaccinated cells of an individual. As used herein, the term"hyperproliferative-associated protein" is meant to refer to proteinsthat are associated with a hyperproliferative disease. To immunizeagainst hyperproliferative diseases, a nucleic acid composition thatincludes a nucleotide sequence which encodes a protein that isassociated with a hyperproliferative disease is administered to anindividual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation genes bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas, and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune diseases.Other tumor-associated-proteins can be used as target proteins, such asproteins which are found at higher levels in tumor cells, including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andbiotechnology, as well as epidemiology, allow for the determination ofprobability and risk assessment for the development of cancer in anindividual. Using genetic screening and/or family health histories, itis possible to predict the probability that a particular individual hasfor developing any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer, or are otherwise in remission,are particularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat such arecurrence. Thus, once it is known that individuals have had a type ofcancer and are at risk of a relapse, they can be immunized in order toprepare their immune systems to combat any future appearance of thecancer.

The present invention also provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of nucleic acid compositions serves as animmunotherapeutic, directing and promoting the immune system of theindividual to combat hyperproliferative cells that produce the targetprotein.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity, including cell receptors and cells which produce"self"-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with a nucleicacid composition that encodes at least one of these proteins will elicitan immune response that will target T cells involved in RA. See: Howell,M. D., et al., 1991 Proc. Natl. Acad. Sci. USA 88:10921-10925; Paliard,X., et al., 1991 Science 253:325-329; Williams, W. V., et al., 1992 J.Clin. Invest. 90:326-333; each of which is incorporated herein byreference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10.Thus, vaccination with a nucleic acid composition that encodes at leastone of these proteins will elicit an immune response that will target Tcells involved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et al., 1990 Nature 345:344-346; eachof which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a nucleic acid composition that encodes atleast one of these proteins will elicit an immune response that willtarget T cells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of such antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes nucleic acidcompositions that encode the variable region of such anti-DNA antibodiesfound in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87:1066, which is incorporated herein by reference.

In aspects of the present invention that relate to gene therapy, thenucleic acid compositions contain either compensating genes or genesthat encode therapeutic proteins. Examples of compensating genes includea gene which encodes dystrophin or a functional fragment, a gene tocompensate for the defective gene in patients suffering from cysticfibrosis, a gene to compensate for the defective gene in patientssuffering from ADA, and a gene encoding Factor VIII. Examples of genesencoding therapeutic proteins include genes which encodeserythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 and TNF.Additionally, nucleic acid compositions which encode single chainantibody components which specifically bind to toxic substances can beadministered. In some preferred embodiments, the dystrophin gene isprovided as part of a mini-gene and used to treat individuals sufferingfrom muscular dystrophy. In some preferred embodiments, a mini-genewhich contains coding sequence for a partial dystrophin protein isprovided. Dystrophin abnormalities are responsible for both the milderBecker's Muscular Dystrophy (BMD) and the severe Duchenne's MuscularDystrophy (DMD). In BMD dystrophin is made, but it is abnormal in eithersize and/or amount. The patient is mild to moderately weak. In DMD noprotein is made and the patient is wheelchair-bound by age 13 andusually dies by age 20. In some patients, particularly those sufferingfrom BMD, partial dystrophin protein produced by expression of amini-gene delivered according to the present invention can provideimproved muscle function.

In some preferred embodiments, genes encoding IL-2, IL-4, interferon orTNF are delivered to tumor cells which are either present or removed andthen reintroduced into an individual. In some embodiments, a geneencoding γ-interferon is administered to an individual suffering frommultiple sclerosis.

Antisense molecules and ribozymes may also be delivered to the cells ofan individual by introducing a nucleic acid composition which acts as atemplate for copies of such active agents. These agents inactivate orotherwise interfere with the expression of genes that encode proteinswhose presence is undesirable. Nucleic acid compositions which containsequences that encode antisense molecules can be used to inhibit orprevent production of proteins within cells. Thus, production ofproteins such as oncogene products can be eliminated or reduced.Similarly, ribozymes can disrupt gene expression by selectivelydestroying messenger RNA before it is translated into protein. In someembodiments, cells are treated according to the invention using nucleicacid compositions that encode antisense or ribozymes as part of atherapeutic regimen which involves administration of other therapeuticsand procedures. Nucleic acid compositions encoding antisense moleculesand ribozymes use similar vectors as those which are used when proteinproduction is desired except that the coding sequence does not contain astart codon to initiate translation of RNA into protein.

Ribozymes are catalytic RNAs which are capable of self-cleavage orcleavage of another RNA molecule. Several different types of ribozymes,such as hammerhead, hairpin, Tetrahymena group I intron, ahead, andRNase P are known in the art; see S. Edgington, Biotechnology (1992) 10,256-262. Hammerhead ribozymes have a catalytic site which has beenmapped to a core of less than 40 nucleotides. Several ribozymes in plantviroids and satellite RNAs share a common secondary structure andcertain conserved nucleotides. Although these ribozymes naturally serveas their own substrate, the enzyme domain can be targeted to another RNAsubstrate through base-pairing with sequences flanking the conservedcleavage site. This ability to custom design ribozymes has allowed themto be used for sequence-specific RNA cleavage; see G. Paolella et al.,EMBO (1992), 1913-1919. ) It will therefore be within the skill of onein the art to use different catalytic sequences from various types; ofribozymes, such as the hammerhead catalytic sequence, and design them inthe manner disclosed herein. Ribozymes can be designed against a varietyof targets including pathogen nucleotide sequences and oncogenicsequences. Preferred embodiments include sufficient complementarity tospecifically target the abl-bcr fusion transcript while maintainingefficiency of the cleavage reaction.

In accordance with the present invention, the multifunctional molecularcomplex containing the desired nucleic acid composition, may beadministered to an individual using a needleless injection device. Inother embodiments, the multifunctional molecular complex containing thedesired nucleic acid composition is simultaneously administered to anindividual intradermally, subcutaneously and intramuscularly using aneedleless injection device. Needleless injection devices are well knownand widely available. One having ordinary skill in the art can,following the teachings herein, use needleless injection devices todeliver multifunctional molecular complexes containing the desirednucleic acid compositions to cells of an individual. Needlelessinjection devices are well suited to deliver these complexes to all ofthese tissues. They are particularly useful to deliver the complexes ofthe present invention to skin and muscle cells.

In some embodiments, a needleless injection device may be used to propelthe complexes of the present invention in liquid form, that contains DNAmolecules, toward the surface of the individual's skin. The liquid ispropelled at a sufficient velocity such that upon impact with the skin,the liquid penetrates the surface of the skin, and permeates the skinand muscle tissue therebeneath. Thus, the nucleic acid composition issimultaneously administered intradermally, subcutaneously andintramuscularly. In some embodiments, a needleless injection device maybe used to deliver nucleic acid compositions to the tissue of otherorgans in order to introduce a nucleic acid molecule to cells of thatorgan.

According to the present invention, the multifunctional molecularcomplexes containing nucleic acid compositions may be administereddirectly into the individual to be immunized or ex vivo into removedcells of the individual which are reimplanted after administration. Byeither route, the genetic material is introduced into cells which arepresent in the body of the individual. Routes of administration include,but are not limited to, intramuscular, intraperitoneal, intradermal,subcutaneous, intravenous, intraarterially, intraoccularly and oral aswell as transdermally or by inhalation or suppository. Preferred routesof administration include intramuscular, intraperitoneal, intradermaland subcutaneous injection. Delivery of nucleic acid compositions whichencode target proteins can confer mucosal immunity in individualsimmunized by a mode of administration in which the material is presentedto tissues associated with mucosal immunity. Thus, in some examples, thenucleic acid composition is delivered by administration to the buccalcavity within the mouth of an individual.

The multifunctional molecular complexes containing nucleic acidcompositions according to the present invention comprise generally fromabout 1 nanogram to about 1000 micrograms of DNA. In some preferredembodiments, the complexes contain about 10 -nanograms to about 800micrograms of DNA. In more preferred embodiments, the complexes containabout 0.1 to about 500 micrograms of DNA. In still more preferredembodiments, the complexes contain about 1 to about 350 micrograms ofDNA. In yet more preferred embodiments, the complexes contain about 25to about 250 micrograms of DNA. In the most preferred embodiments, thecomplexes contain about 100 micrograms DNA.

The multifunctional molecular complexes containing nucleic acidcompositions according to the present invention are formulated accordingto the mode of administration to be used. One having ordinary skill inthe art can readily formulate a pharmaceutical composition thatcomprises a nucleic acid composition. In cases where intramuscularinjection is the chosen mode of administration, an isotonic formulationis preferably used. Generally, additives for isotonicity can includesodium chloride, dextrose, mannitol, sorbitol and lactose. In somecases, isotonic solutions such as phosphate buffered saline arepreferred. Stabilizers include gelatin and albumin. In some embodiments,a vasoconstriction agent is added to the formulation. The pharmaceuticalpreparations according to the present invention are prepared so as to besterile and pyrogen free.

In addition to other agents which may function as transfecting agentsand/or replicating agents, there may be co-administered with thecomplexes of the present invention growth factors, cytokines andlymphokines such as α-interferon, 7-interferon, platelet derived growthfactor (PDGF), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1,IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, as well as fibroblast growthfactor, surface active agents such as immune-stimulating complexes(ISCOMS), Freund's incomplete adjuvant, LPS analog includingmonophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs andvesicles such as squalene and hyaluronic acid may also be used,administered in conjunction with the complexes of the present invention.In some embodiments, combinations of these agents are administered inconjunction with the complexes of the present invention.

The complexes of the present invention may be combined with collagen asan emulsion and delivered parenterally. The collagen emulsion provides ameans for sustained release of DNA; 50 μl to 2 ml of collagen may beused. About 100 μg of DNA are combined with 1 ml of collagen in apreferred embodiment using this formulation. Other sustained releaseformulations such as those described in Remington's PharmaceuticalSciences, A. Osol, a standard reference text in this field, which isincorporated herein by reference. Such formulations include aqueoussuspensions, oil solutions and suspensions, emulsions and implants aswell as reservoirs, depots and transdermal devices. In some embodiments,time release formulations for the complexes are preferred; where it isdesirable the complex be time released between 6-144 hours, preferably12-96 hours, more preferably 18-72 hours.

In some embodiments of the invention, the individual is subject to asingle vaccination to produce a full, broad immune response. In otherembodiments of the invention, the individual is subject to a series ofvaccinations to produce a full, broad immune response. According tostill other embodiments of the invention, at least two and preferablyfour to five injections are given over a period of time. The period oftime between injections may be from 24 hours apart to two weeks orlonger between injections, preferably one week apart. Alternatively, atleast two and up to four separate injections are given simultaneously atdifferent sites.

In some embodiments of the invention, a complete vaccination includesinjection of a single inoculant which contains a nucleic acidcomposition including sequences encoding one or more targeted epitopes.

In other embodiments of the invention, a complete vaccination includesinjection of two or more different inoculants into different sites. Forexample, an HIV vaccine may comprise two different inoculants in whicheach one comprises a nucleic acid composition encoding different viralproteins. This method of vaccination allows the introduction of as muchas a complete set of viral genes into the individual without the risk ofassembling an infectious viral particle. Thus, an immune responseagainst most or all of the virus can be invoked in the vaccinatedindividual. Injection of each inoculant is performed at different sites,preferably at a distance to ensure that no cells receive the totalcombination of nucleic acid compositions. As a further safetyprecaution, some genes may be deleted or altered to further prevent thecapability of infectious viral assembly.

In accordance with the present invention there are providedpharmaceutical compositions which facilitate delivery of themultifunctional molecular complex, which in turn functions to facilitatetransfer of the nucleic acid composition which is contained therein, tothe target cells. The pharmaceutical composition may be nothing morethan an inert diluent and a pharmaceutically acceptable salt or esterform of said molecular complex. However, other pharmaceuticallyacceptable carriers well known to the artisan in this field, can also besuitably employed to provide desired properties. Thus, one or moreagents may be selected from the following recognized pharmaceuticalclasses of excipients: solvents, solvent systems, and solubilizing anddispersing agents including surfactants and emulsifying agents;viscosity modifying agents; and stabilizing and preservative agents,including antioxidants, WV absorbing agents, antibacterial agents, andbuffering agents.

The present invention also provides pharmaceutical kits which comprise acontainer comprising a nucleic acid composition, and a containercomprising a transfer moiety. Optionally, there is included in such kitsexcipients, carriers, preservatives and vehicles of the type describedabove with respect to pharmaceutical compositions. The termpharmaceutical kit is also intended to include multiple inoculants usedin the methods of the present invention. Such kits include separatecontainers comprising different inoculants and transfer moieties. Thepharmaceutical kits in accordance with the present invention are alsocontemplated to include a set of inoculants used in immunizing methodsand/or therapeutic methods, as described above.

The compositions and methods of the present invention are useful in thefields of both human and veterinary medicine. Accordingly, the presentinvention relates to genetic immunization and therapeutic treatment ofmammals, birds and fish. The methods of the present invention can beparticularly useful for genetic immunization and therapeutic treatmentof mammalian species including human, bovine, ovine, porcine, equine,canine and feline species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Examples set out below include representative demonstrations ofvarious aspects of the present invention. The Examples are not intendedto limit the scope of the invention; but rather are merely intended toserve as illustrations thereof. Moreover, one having ordinary skill inthis art will be able readily to appreciate additional aspects andembodiments of the present invention, based on the foregoing detaileddescription thereof. Unless otherwise indicated, all temperaturesrecited in the following Examples are Celsius scale temperatures.

EXAMPLE 1 Preparation of N⁴ -5'-Aminopentylspermidine Hydrochloride 8

N⁴ -(4-cyanobutyl)-N¹,N-⁶ (tert -butyloxycarbonyl) -Spermidine (6)

A solution of N¹,N⁸ -Bis(tert-butyloxycarbonyl) spermidine (2.92 g, 8.45mmol, 1.0 eq) S. Nagarajan and B. Ganem, J. Org. Chem., 50, 5735-37(1985)! in acetonitrile (125 mL) was treated withN,N-diisopropylethylamine (3.534 mL, 20.0 mmol, 2.4 eq), potassiumiodide (2.81 g, 16.90 mmol, 2.0 eq), and 5-chlorovaleronitrile (1.902mL, 16.90 mmol, 2.0 eq). The resulting homogeneous solution was heatedto reflux for 2 hours. The mixture was treated with additionalN,N-diisopropylethylamine (1.767 mL; 1.2 eq), potassium iodide (1.41g,1.0 eq) and 5-chlorovaleronitrile (0.951 mL, 1.0 eq), and refluxed anadditional 18 hours. Thin layer chromatography (TLC) indicated noremaining starting material. The acetonitrile was removed under vacuum,and the residue taken up in chloroform (250 mL). This solution waswashed with water (200 mL), dried (Na₂ SO₄), and stripped of solvent toafford crude product as an oil. The material was purified on silicausing a gradient of 2-propanol in chloroform plus 1%N,N-diisopropylethylamine to give an oil (3.40 g); ¹ H NMR (CDCl₃): δ1.44 (s, 20.8 H; should be 18 H), 1.58-1.80 (m, 9.8 H), 2.38-2.44 (m,9.4 H; should be 8.0 H), 3.16 (m, 4.1 H), 4.80 (m, 0.8 H), 5.38 (m, 0.8H).

N⁴ (5-aminopentyl) -N¹ ₁,N⁷ Bis(tert-butyloxycarbonyl)-Spermidine (7)

A solution of 6(0.77g, 1.81 mmol) in glacial acetic acid (100 mL) wastreated with 5-palladium on carbon (0.08 g, 10% w/w) and placed on aParr Hydrogenator (50 psi hydrogen gas pressure) for 2.75 hours. Themixture was filtered through Celiteo brand diatomaceous earth filter aid(pre-rinsed with glacial acetic acid) and the Celite rinsed withchloroform. The filtrate and chloroform wash were combined and solventremoved to give an oil. The crude material was subjected to silicachromatography using a gradient of methanol in chloroform plus 0.4%diisopropylethylamine. The material consisted of a colorless oil (0.32g). ¹ H NMR (CDCl₃): δ 1.33 (m, 3.5 H; should be 2.0 H), 1.44 (s, 19.1H; should be 18.0 H), 1.59 (m, 10.3 H), 2.37-2.45 (m, 6.2 H), 2.70 (t,1.5 H), 3.14 (m, 4.0 H), 4.89 (m, 0.7 H), 5.57 (m, 0.8 H).

N⁴ -(5 -Aminopentyl)spermidine Hydrochloride (8)

Compound 7 (0.200 g, 0.46 mmol) was stirred with trifluoroacetic acid (5mL) at room temperature for 2 hours. The trifluoroacetic acid wasremoved under vacuum, followed by three chloroform additions andsubsequent evaporations under vacuum. The resulting crude oil was twicetaken up in 0.1N HCl (30 mL) and lyophilized to give 8 as a hydroscopicsolid (0.19 g).

EXAMPLE 2 Preparation of N⁴ -(5-(β-3'-propionylgalactosyl-β1"-4'-thioglucoside)aminopentyl)spermidine 9

S-(Succinimidyl-β-3'-propionyl)hepta-O-acetylgalactosyl-α1'4-thioglucoside (10)

A solution of S-β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside M. Elofsson, S. Roy, B. Walse and J.Kihlberg, Carb. Res., 246, 89-103 (1993)! (4.60 g, 6.35 mmol) in 1:1isopropanol/chloroform (100 mL) was treated with N-hydroxysuccinimide(0.73 g, 6.35 mmol) and N, N'-dicyclohexylcarbodiimide (1.31 g, 6.35mmol). After stirring at room temperature for 19 hours, the mixture wascooled to 4° for 1 h and filtered. The solvent was removed from thefiltrate under vacuum to give a white solid that was recrystallized from2-propanol (3.43 g). The isolated product was 92% pure by highperformance liquid chromatography (HPLC). ¹ H NMR (CDCL₃): δ 2.0-2.16(7s, 21.0 H), 2.85 (s, 3.8 H), 2.85-3.1 (m, 4.2 H), 3.65 (m, 0.7 H),3.78 (t, 1.0 H), 3.88 (t, 1.0 H), 4.11 (m, 4.0 H), 4.54 (m, 2.8 H), 4.97(m, 1.81 H), 5.11 (m, 1.0 H), 5.23 (t, 1.0 H), 5.36 (d, 0.7 H).

N'(5(S-β-3'-propionyl hepta-O-acetylgalactosyl-β1"-4'-thioglucoside)aminopentyl)-N¹,N⁶-bis(tert-butyloxycarbonyl)-Spermidine (11)

A solution of 10 (0.300 g, 0.70 mmol) in methylene chloride (30 mL) wastreated with a solution of 7 (0.57 g, 0.70 mmol) in methylene chloride(30 mL). The mixture was stirred at room temperature for 18 hours. Thesolvents were removed under vacuum. Silica chromatography using agradient of 2-propanol in chloroform afforded purified product as acolorless glass (0.49 g); purity of 100% as determined by HPLC. ¹ H NMR(CDCL₃): δ 1.33 (m, 3.0 H; should be 2.0 H), 1.44 (s, 20.0 H; should be18.0 H), 1.54 (m, 10.7 H; should be 6.0 H), 1.68 (m, 2.0 H), 1.97-2.16(7s, 27.3 H; should be 21.0 H), 2.25 (m, 6.0 H; should be 4 H), 2.52 (m,9.0 H; should be 8.0 H), 2.84 (m, 1.0 H), 3.01 (m, 1.0 H), 3.1-3.27 (m,6.7 H), 3.64 (m, 1.0 H), 3.80 (t, 1.0 H), 3.90 (t, 1.0 H), 4.11 (m, 4.0H), 4.55 (d, 2.0 H), 4.70 (d, 1.0 H), 4.91-5.0 (m, 3.0 H; should be 2.0H), 5.11 (m, 1.3 H), 5.21 (t, 1.0 H), 5.36 (d, 1.0 H), 5.44 (m, 0.7 H),6.28 (m, 0.7 H). FAB Mass Spec MH⁼ 1138.

N⁴ -(5-(S-β-3'-propionyl hepta-O-acetylgalactosyl-β1"-4'-thioglucoside)aminopentyl)spermidine trifluoroacetate(12)

Compound 11 (0. 200 g, 0.18 mmol) was treated with trifluoroacetic acid(5 mL) and stirred at room temperature for 2 hrs. The trifluoroaceticacid (TFA) was largely removed under vacuum, and the residue subjectedto three additions of chloroform, followed by removal of solvent undervacuum. The product was recovered as an oil (0.22 g) with trace TFA inevidence; purity of 100% as determined by HPLC. ¹ H NMR (CDCL₃): δ1.25-1.53 (m, 5.5 H), 1.6-2.0 (m, 5.5; should be 4 H), 2.00-2.15 (7s,23.0 H; should be 21.0 H), 2.56 (m, 1.0 H), 2.68 (m, 1.3 H), 2.80 (m,1.0 H), 3.0-3.4 (m, 11.OH; should be 10.0 H), 3.64 (m, 1.0 H), 3.79 (m,1.0 H), 3.94 (m, 1.0 H), 4.11 (m, 2.5 H), 4.55 (m, 1.5 H), 4.70 (m, 1.3H), 4.90-5.20 (broad m, 16.0 H; inflated by water; should be 2.0 or 4.0H), 5.36 (m, 1.0 H), 7.12 (m, 0.5 H), 7.86 (m, 2.3 H), 8.07 (m, 2. 3H),9. 8 (m. 0.5 H).

N⁴ -(5-(β-3'-propionylgalactosyl-β1"-4'-thioglucoside)-aminopentyl)spermidine (9)

A solution of 12 (0.20 g; 0.18 mmol) in methanol (20 mL) was treatedwith sodium carbonate (0.38 g; 3.08 mmol; 18 eq) and water (35 mL) for ahomogeneous solution. After 6 hrs at room temperature, the solvents wereevaporated and the residue was desalted using Sephadex G-25 Medium gelfiltration resin, and 1% glacial acetic acid as eluant. Fractionscontaining product were combined and lyophilized for pure product as thetriacetate salt (0.10 g); purity of 100% as determined by HPLC. ¹ H NMR(DMSO- d₆ D₂ O): δ 1.15 (m or broad t, 1.5 H), 1.3-1.5 (m, 8.0 H), 1.65(m, 2.0 H), 1.66 (s, 14.0 H; should be 9.0 H), 2.34 (m, 6.5 H), 2.60 (m,3.0 H; should be 2.0 H), 2.74 (m, 6.0 H), 3.00 (m, 3.0 H), 3.27 (m, 3.5H), 3.40 (m, 1.5 H), 3.47 (m, 2.0 H), 3.50 (m, 1.0 H), 3.60 (m, 1.0 H),3.71 (d, 1.0 H), 4.14 (broad s, masked by water peak), 4.27 (m, 4.OH).

EXAMPLE 3 Preparation of N⁴ -(5- N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ (β-3'-propionylgalalactosyI-β1-4-thioglucoside)lysyl!-amino)pentylspermidine acetatesalt 18

For comparative purposes, the CAS style name of the compound 18 set outabove is as follows: 4- (N² - N²,N⁶ -bis(3-4-O(β-D-galactopyranosyl)-(β-D-glucopyranosylthio!propionyl)lysyl!N⁶-(3-4-O-(β-D-galactopyranosyl)-β-D-glucopyranosylthio!-propionyl)lysinamido)pentyl!-1,8-diamino-4-azaoctane,acetate salt.

N⁴ -(5- N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl N⁶ -(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside) lysine (14)

To a solution of lysyl-lysine (0.25 g, 0.64 mmol) in 1:1water/acetonitrile (100 mLs) was added N,N-diisopropylethylamine (0.336mls, 1.95 mmol, 3.0 eq) and compound 10 (1.859, 2.25 mmol) and stirredat room temperature for a few minutes until homogeneity was achieved.The pH was closely monitored at regular intervals, andN,N-diisopropylethylamine added as needed to maintain the pH between 7and 8. A total of 7 eq of base was added over 1 hour before the pHstabilized at 7-7.5. Reverse phase HPLC was used to follow the progressof the reaction. After 24 hrs at room temperature, the reaction appearedto have stopped with approximately 50% product formation. Theacetonitrile was evaporated under vacuum, and the aqueous mixturetreated with dilute HCl (pH 5). The solution was extracted intochloroform (2×200 mls). The combined organic layers were dried (Na₂SO₄), and the solvent removed under vacuum to afford crude product as aglass (2.17 g). The material was subjected to silica flashchromatography using a gradient of isopropanol in chloroform plus 1%glacial acetic acid as eluant (1.09 g). Purity was determined by HPLC tobe approximately 96%. FAD Mass Spec: MH+=2394.

Succinimidyl N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysine (15)

A solution of N², N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysine (1.00 g, 0.42 mmol) in 1:1isopropanol/chloroform (20 mL) was treated with N-hydroxysuccinimide(48.1 mg, 0.42 mmol) and N,N' dicyclohexylcarbodiimide (86.2 mg, 0.42mmol). After stirring at room temperature for 19 hours, the mixture wascooled to 4° for 1 h and filtered. Solvent was removed from the filtrateunder vacuum, and the crude product was recrystallized from 2-propanol.The product was collected by filtration and dried under vacuum to give awhite powdery solid (0.76 g). This material was shown by HPLC to consistof a mixture of the starting free acid and the succinimidyl ester in aratio of approximately 1:2 respectively. The mixture was not subjectedto further purification, but was used as is in the next step. ¹ H NMR(CDCl₃): δ 1.96-2.20 (multiple s, 86.4 H; should be 63.0 H), 2.28 (m,3.6 H), 2.53 (m, 6.6 H), 2.88 (m, 6.6 H), 3.02 (m, 3.6 H), 3.18-3.40 (m,4.2 H), 3.62 (m, 3.6 H), 3.85 (m, 3.6 H), 3.95 (m, 3.6 H), 4.10 (m, 12.0H), 4.57 (m, 6.0 H), 4.70 (m, 4.2 H), 5.02 (m, 7.2 H), 5.10 (m, 3.6 H),5.20 (t, 3.6 H), 5.36 (d, 3.0 H), 6.31 (t, 0.6 H), 6.58 (t, 0.6 H), 6.90(d, 0.6 H), 7.47 (d, 0.6 H).

N⁴ -(5- N² N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl!-aminopentyl-N¹,N⁶-Bis(tert-butoxycarbonyl)spermidine (16)

A solution of 7 (87 mg, 0.20 mmol) in methylene chloride (20 mL) wastreated with a solution of 15 (0.76 g of the 660 mixture, correspondingto 0.50 g of the ester; 0.20 mmol) in methylene chloride (20 mL). Themixture was stirred at room temperature for 18 hrs, followed by removalof the solvent under vacuum. The crude product was purified by silicachromatography using a gradient of isopropanol in chloroform. Theresulting impure product consisted of a 65:35 mixture of product to thefree acid present as the contaminant in the starting ester. A secondsilica column using the same gradient plus 0.5% glacial acetic acideffectively isolated pure product (0.38 g); purity of 100% as determinedby HPLC. ¹ H NMR (CDCl₃): δ 1.30-1.39 (m, 4.0 H), 1.44 (s, 13.5 H;should be 18.0 H), 1.53 (m, 8.6H), 1.60-1.90 (m, 6.1 H), 1.97-2.16(multiple s, 73.8 H; should be 63.0 H), 2.27 (m, 3.7 H), 2.49-2.70 (m,8.0 H), 2.85 (m, 2.5 H), 3.00 (m, 2.5 H), 3.23 (m, 6.2 H), 3.65 (m, 2.5H), 3.80-3.93 (m, 5.5 H), 4.12 (m, 8.0 H), 4.20-4.40 (m, 1.8 H),4.57-4.69 (m, 8.0 H), 4.91-5.00 (m, 5.5 H), 5.09(m, 3.1 H), 5.21 (t, 3.1H), 5.36 (d, 2.5 H), 6.57 (m, 0.9 H), 6.95 (m, 0.6 H), 7.20 (m, 0.6 H).

N⁴ -(5- N², N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl!-aminopentyl)spermidine (17)

Compound 16 (0.170 g, 0.059 mmol) was treated with trifluoroacetic acid(5 mL) and stirred at room temperature for 2.5 hours. Thetrifluoroacetic acid was largely removed under vacuum (0.19 g). Puritywas determined by HPLC to be approximately 100%. ¹ H NMR (CDCl₃): δ1.20-1.80 (m, 4.8 H), 1.96-2.15 (multiple s, 84.0 H; should be 63.0 H),2.54 (m, 12.0 H; possibly inflated by water peak), 3.65 (m, 3.0 H), 3.81(m, 3.0 H), 3.93 (m, 4.0 H), 4.12 (m, 12.0 H), 4.29 (m, 2.0 H), 4.55 (m,6,0 H), 4.69 (m, 4.0 H), 4.89 (m, 3.0 H), 5.09 (m, 6.0 H), 5.19 (m, 4.0H), 5.38 (d, 3.0 H), 6.99 (m, 1.0 H), 7.60 (m, 0.5 H), 7.91 (m, 1.0 H),8.09 (m, 1.0 H).

N⁴ -(5- N²,N⁶ -bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysy!-aminopentyl)spermidine acetate salt(18)

To a solution of 17 (0.17 g, 0.059 mmol) in methanol (20 mL) was addedsodium carbonate (0.37 g, 2.95 mmol), followed by water (40 mL) untilhomogeneity was achieved. After stirring at room temperature for 4 hrs,the solvents were stripped off and the crude product eluted down aSephadex G-25 Medium column using 1% glacial acetic acid as the eluant.Fractions containing product were combined and lyophilized to affordpure product as an extremely hygroscopic solid (0.07 g).

EXAMPLE 4 Preparation of N⁴ -(5- N² -N⁶ -bis(β-3'-proplonylgalactosyl-β1-4-thioglucoside)lysyl!-aminopentyl)spermidine acetate salt23

Succinimidyl N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysine (20)

A solution of N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysine (1.00 g, 0.42 mmol) in 1:1isopropanol/chloroform (20 mL) is treated with N-hydroxysuccinimide(48.1 mg, 0.42 mmol) and N,N' dicyclohexylcarbodiimide (86.2 mg, 0.42mmol). After stirring at room temperature for 19 hours, the mixture iscooled to 40 for 1 h and filtered. Solvent is removed from the filtrateunder vacuum, and the crude product recrystallized from 2-propanol. Theproduct is collected by filtration and dried under vacuum to give awhite powdery solid (0.76 g). This material is shown by HPLC to consistof a mixture of the starting free acid and the succinimidyl ester in aratio of approximately 1:2 respectively. The mixture is not subjected tofurther purification, but is used as is in the next step.

N⁴ -(5- N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)-N¹,N⁶-Bis(tert-butoxycarbonyl)spermidine (21)

A solution of 7 (87 mg, 0.20 mmol) in methylene chloride (20 mL) istreated with a solution of 20 (0.769 of the 66% mixture, correspondingto 0.50 g of the ester; 0.20 mmol) in methylene chloride (20 mL). Themixture is stirred at room temperature for 18 hrs, followed by removalof the solvent under vacuum. The crude product is purified by silicachromatography using a gradient of isopropanol in chloroform. Theresulting impure product consists of a 65:35 mixture of product to thefree acid present as the contaminant in the starting ester. A secondsilica column using the same gradient plus 0.5% glacial acetic acideffectively isolates pure product (0.38 g); purity of 100% as determinedby HPLC.

N⁴ -(5- N²,N⁶ -bis(β-3'-propionyl hepta-O-acetylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)spermidine (22)

Compound 21 (0.170 g, 0.059 mmol) is treated with trifluoroacetic acid(5mL) and stirred at room temperature for 2.5 hours. The trifluoroaceticacid is largely removed under vacuum (0.19 g). Purity is determined byHPLC to be approximately 100%.

N⁴ -(5- N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)spermidine acetate salt(23)

To a solution of 22 (0.17 g, 0.059 mmol) in methanol (20 mL) is addedsodium carbonate (0.37 g, 2.95 mmol), followed by water (40 mL) untilhomogeneity is achieved. After stirring at room temperature for 4 hrs,the solvents are stripped off and the crude product is eluted down aSephadex G-25 Medium column using 1 glacial acetic acid as the eluant.Fractions containing product are combined and lyophilized to afford pureproduct as an extremely hydroscopic solid (0.07 g).

EXAMPLE 5 Preparation of N⁴ -5-(D-biotinyl)aminopentyl spermidinehydrochloride salt 24

To a solution of 7 (0.20 g, 0.47 mmol) in 20 mL of acetonitrile and 10mL of water was added 160 mg of succinimidyl D-biotin. The solution wasstirred for 18 h. The volume of the solution was reduced to 15 mL undervacuum and the remaining solution was purified on octadecylsilyl bondedsilica using a water/acetonitrile gradient containing0.1-trifluoroacetic acid. Fractions containing the product were combinedand solvent removed under vacuum to give a waxy white solid (94% pure byHPLC). To the white solid was added 10 mL of 2-propanol and 10 mL of 4 NHCl in dioxane. The solvents were removed under vacuum. The resultantwhite solid was redissolved in water and dried under vacuum to give ahygroscopic foam (0.11 g). ¹ H NMR (DMSO-d₆) δ 1-31 (m, 4 H), 1.44 (m, 6H), 1.64 (m, 8 H), 2.09 (m, 4 H), 2.63 (d, 2 H), 2.91 (m, 4 H), 3.06 (m,8 H), 4.18 (m, 1 H), 4.36 (m, 1 H).

EXAMPLE 6 Preparation of N⁴ -(5-cholestene-3'β-oxycarbonyl)aminopentylspermidine hydrochloride salt 25

To a solution of 7 (0.20 g, 0.47 mmol) in 20 mL of methylene chloridewas added 210 mg of cholesteryl chloroformate and 200 μL ofdiisopropylethylamine. The solution was stirred for 24 h. The methylenechloride was removed under vacuum and the remaining oil was redissolvedin chloroform and purified on silica using a methanol/chloroformgradient containing 0.1% diisopropylethylamine. Fractions containing theproduct were combined and solvent removed under vacuum to give a waxywhite solid (0.29 g). To the white solid was added 10 mL of 2-propanoland 10 mL of 4 N HCl in dioxane. The solvents were removed under vacuum.The resultant white solid was redissolved in water and dried undervacuum to give a waxy white solid (0.15 g).

EXAMPLE 7 Preparation of N⁴ -octyl-N¹,N⁸ -Bis(tert-butyloxycarbonyl)spermidine 26

To a solution of N¹,N⁸ -Bis(tert-butyloxycarbonyl) spermidine (1.0 g,2.89 mmol) in acetone (100 mls) was added N,N-diisopropylethylamine(0.605 mls, 3.47 mmol, 1.2 eq), potassium iodide (0.489, 2.89 mmol), and1-bromooctane (0.500 mls, 2.89 mmol). The mixture was heated to refluxfor one hour, followed by the addition of N,N-diisopropylethylamine(0.605 mls, 3.47 mmol) and potassium iodide (0.48 g, 2.89 mmol). Afteran additional 3 hrs reflux, 1-bromooctane (0.500 mls, 2.89 mmol) wasadded and refluxing continued for an additional hour. The acetone wasevaporated under vacuum. The residue taken up in chloroform (125 mls)and washed with water (2×75 mls). The organic layer was dried (Na2 504),and solvent removed under vacuum to give a liquid. The liquid waspurified by silica chromatography using a gradient of isopropanol inchloroform plus 1% N,N-diisopropylethylamine. The pure product wasrecovered as a pale orange oil (0.59 g). ¹ H NMR (CDCl3): δ 0.88 (t, 3.5H), 1.27 (m, 14.2 H; should be 16.0 H), 1.44 (s, 18.7 H), 1.58 (m, 1.6H), 1.82 (m, 1.6 H), 2.37 (m, 2.4 H), 2.44 (m, 2.2 H), 3.19 (m, 4.0 H),4.84 (m, 0.3 H), 5.62 (m, 0.3 H).

The N⁴ -octyl-N¹,N⁸ -Bis(tert-butyloxycarbonyl) spermidine (0.15 g) wasdissolved in 6 mL of 4N HCl in dioxane and stirred at room temperaturefor 1 h. The solvent was removed under vacuum and the yellow oilsuspended in chloroform and the solvent removed under vacuum. Theresultant oil was dissolved in 10 mL of absolute ethanol andprecipitated by the addition of 30 mL of diethyl ether. The solid wasisolated by decanting off the liquid and drying under vacuum (0.10 g). ¹H NMR (DMSO- d₆): δ 0.86 (m, 3 H), 1.28 (m, 10 H), 1.58-1.80 (m, 6 H),2.00 (m, 2 H), 2.80 (m, 2 H), 2.89 (m, 2 H), 3.03 (m, 4 H), 3.15 (m, 2H), 8.01 and 8.13 (two m, 5 H, should be 6 H). Anal: Calcd for C₁₅ H₃₈Cl₃ N₃ C 49.11, H 10.44, N 11.45. Found C 48.57, H10.75, N 11.29.

EXAMPLE 8 Preparation of N-dodecylspermidine trihydrochloride 27

A solution of N¹,N⁸ -Bis(tert-butyloxycarbonyl) spermidine (0.50g, 1.45mmol) in acetone (50 mLs) was treated with N,N-diisopropylethylamine(0.604 mLs, 3.48 mmol), potassium iodide (0.48 g, 2.90 mmol), and1-bromododecane (0.348 mLs, 2.90 mmol). The mixture was refluxed for 18hrs and was treated with additional 1-bromododecane (0.348 mLs, 2.90mmol). Refluxing was continued for 4 hours. The acetone was removedunder reduced pressure, and the residue taken up in chloroform (250mLs). The solution was washed with water (2×100 mLs). The organic layerwas dried (Na₂ S)₄), and the solvent removed for crude product. Thematerial was purified by silica flash chromatography eluting with agradient of isopropanol in chloroform plus 0.4%N,N-diisopropylethylamine. The pure product was recovered as an oil ofmass 0.52 g. ¹ H NMR (CDCl₃): δ 0.88 (t, 3.0 H), 1.26 (broad s, 21.0 H;should be 20.0 H), 1.44 (s, 20.5; should be 18.0 H), 1.64 (m, 5.8 H),2.37 (m, 3.8 H), 2.46 (t, 2.5 H), 3.15 (m, 4.0 H), 4.84 (m, 0.5 H), 5.62(m, 0.8 H).

The N¹,N⁸ -Bis (tert-butyloxycarbonyl) -N4-dodecyl-spermidine (0.52g,1.01 mmol) was dissolved in 2-propanol (5 mLs) and treated with 4N HClin dioxane (10 mLs). The homogeneous solution was stirred at roomtemperature for 20 hrs, and the solvents were evaporated under reducedpressure. The crude oil was taken up in ethanol (20 mLs) and treatedwith ether (10-15 mLs) with stirring. A solid precipitated out ofsolution and was collected by filtration (85 mgs). A second crop wasrecovered for an additional 79 mgs. Total yield was 164 mgs. ¹ H NMR(DMSO-d₆): δ 0.86 (t, 2.3 H), 1.25 (m, 18.0 H), 1.63 (m, 3.7 H), 1.76(m, 2.3 H), 1.99 (m, 1.3 H), 2.79 (m, 2.0 H), 2.90 (m, 2.0 H), 3.02 (m,3.7 H), 3.16 (m, 2.3 H), 8.03 (m, 1.7 H), 8.14 (m, 2.7 H).

EXAMPLE 9 Preparation of N-hexadecylspermidine trihydrochloride 28

A solution of N¹,N⁸ -Bis(tert-butyloxycarbonyl) spermidine (0.50 g, 1.45mmol) in acetone (50 mLs) was treated with N,N-diisopropylethylamine(0.302 mLs, 1.74 mmol, 1.2 eq), potassium iodide (0.24 g, 1.45 mol) and1 bromohexadecane (0.442 mLs, 1.45 mmol). The mixture refluxed for 20hrs, and the solvents were removed under vacuum. The residue was takenup in chloroform (250 mLs) and washed with water (150 mLs). The organiclayer was dried (Na₂ SO₄), and the solvent evaporated under reducedpressure to afford crude product as an amber oil. The material waspurified by silica flash chromatography using a gradient of isopropanolin chloroform plus 0.4% N,N-diisopropylethylamine. The pure product wasrecovered as a pale yellow oil, mass 0.42 g. ¹ H NMR (CDCl₃): δ 0.88 (t,2.9 H), 1.26 (broad s, 30.3 H; should be 28.0 H), 1.44 (s, 22.9 H;should be 18.0 H), 1.60 (m, 4.0 H), 1.72 (m, 1.7 H), 2.37 (m, 3.7 H),2.46 (m, 2.9 H; should be 2.0 H), 3.17 (m, 4.0 H), 4.83 (m, 0.6 H), 5.62(m, 0.6 H).

The N¹,N⁸ -Bis(tert-butyloxycarbonyl)-N⁴ -hexadecyl-spermidine (0.42 g,0.74 mmol), was dissolved in 2-propanol (5 mLs) and was treated with 4NHCl in dioxane (10 mLs). The homogeneous solution was stirred at roomtemperature for 1.5 hrs, followed by evaporation of the solvents underreduced pressure. The residue was taken up in ethanol (12 mLs) and wastreated with ether (10 mLs) with stirring. A precipitate fell out ofsolution and was collected by filtration; mass 125 mgs. A second cropwas recovered of mass 75 mgs for a total yield of 0.200 g. ¹ H NMR (DMSO-d₆ +D₂ O): δ 0.83 (t, 2.1 H; should be 3.0 H), 1.22 (m, 26.6 H),1.54-1.70 (m, 6.0 H), 1.96 (m, 1.7 H), 2.81 (m, 1.7 H), 2.89 (m, 2.1 H),3.04-3.12 (m, 6.0 H).

EXAMPLE 10 Transfection of Adherent Cells In Culture Using ReceptorBearing Cells

Human hepatocellular carcinoma HuH7 cells were grown and seeded into a96 well plate with 1-2×10⁴ cells in 100 μL of minimal essential media α,modification with 10% serum. The plates were incubated in a 37° CO₂incubator until the cells were 60-80 % confluent (approximately 24 hrs).The media was removed and the cells washed once with Optimem® (serumfree media). Optimem® (100 μL) containing 0.5 μg of pCMVβ plasmid (fromClonetech, No. 6177-1), 0.5 μg of N⁴ -(5- N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)-spermidine and 2.5 μgof N⁴ -(5-cholestene-3,'β-oxycarbonyl)-aminopentyl)spermidine was addedto the cells. The cells were incubated with the mixture for 4 hrs in a37° CO₂ incubator; thereafter 100 μL of media containing 20% serum wasadded and the incubation continued for an additional 20 hrs. Cells lysedwith 0.5% NP-40 in 140 mM NaCl, 10 mM tris, 1.5 mM MgCl₂ were assayedfor β-galactosidase activity using o-nitrophenyl β-D-galactopyranoside,and gave A₄₀₅ =1.35 after 30 min. Cells grown in 6 well plates (25 mmdiameter) and treated similarly were fixed with 2% paraformaldehyde andassayed for β-galactosidase activity using 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (J. Sambrook, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, 1989). Visual inspection showed5-10% of the cells were transfected as evidenced by light microscopy.Similar results were obtained when the receptor specific bindingcomponent of the multifunctional molecular complex was omitted from thecell culture, while retaining the endosome membrane disruption promotingcomponent of the multifunctional molecular complex.

Similar results were obtained with N⁴ -(5-(β-3'-propionylgalactosyl-β1-4-thioglucoside)aminopentyl) spermidine; and N⁴-(5-(methyltetrahydrofolyl)aminopentyl)-spermidine; with the endosomemembrane disruption promoting components included therein. Similarresults can also be obtained with N⁴ -octylspermidine; N⁴-dodecylspermidine; fusogenic peptides acylated on the N-terminus by N⁴-(5-carboxypentyl)spermidine; N⁴-(5-(3α,7α,12α-trihydroxy-5β-cholan-24-oic)aminopentyl) spermidineamide, with the receptor specific binding components included therein.

EXAMPLE 11 Transfection of Muscle Cells In Vivo

Solutions were prepared containing 100 μg of pCMVβ plasmid and 100 μg ofeither N⁴ -octylspermidine, N⁴ -dodecylspermidine or N⁴-(5-cholestene-3'β-oxycarbonyl)-aminopentyl)spermidine, each in 100 μLof phosphate buffered saline. The plasmid solution (100 μL) was injectedinto the rear quadriceps of 6-12 week old BALB-C mice. The mice weresacrificed approximately 96 hrs later and the entire quadriceps muscletissue was removed. The muscle was fixed with formalin for 2 hrs, andassayed for β-galactosidase activity using5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (1 mg/mL in tris/EDTApH 8.5). All three injections were scored as positive since the bluecolor was more intense than the control: injection of 100 μg of pCMVβplasmid injected in 100 μL of 0.25% bupivicaine hydrochloride in citratebuffer pH 6.0. Similar results can also be obtained with fusogenicpeptides acylated on the N-terminus by N⁴ -(5-carboxypentyl) spermidine;and N⁴ -(5-(3α,7β,12β-trihydroxy-5β-cholan-24-oic)aminopentyl)spermidine amide.

EXAMPLE 12 Transfection of Liver Cells In Vivo

A solution is prepared containing 10 μg of PHBVSA plasmid, 5 μg of N⁴-(5- N²,N⁶ -bis(β-3'-propionyl galactosyl-β1-4-thioglucoside) lysyl-N⁶-(β3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)spermidine and 25 μg ofN⁴ -(5-cholestene-3'β-oxycarbonyl)aminopentyl)spermidine in 100 μL ofphosphate buffered saline. The plasmid solution (100 μL) is injectedinto the tail vein of 6-12 week old BALD-C mice. The mice are sacrificed48-120 hrs later and the serum tested for hepatitus P surface antigenusing a commercial enzyme-linked immunoassay. The production of surfaceantigen is greater than the positive control supplied with the kit at 48hrs post-injection.

Similar results are obtained with N⁴ -(5-(β3'-propionylgalactosyl-β1-4-thioglucoside)aminopentyl)spermidine; N² N⁶ -(5-bis(β3'-propionyl galactosyl-β1-4-thioglucoside)lysyl! aminopentyl)spermidine; N⁴ -(5-(methyltetrahydrofolyl) aminopentyl)spermidine; N⁴-(5-(folinyl)aminopentyl)-spermidine; N⁴ -(5-(α-3'-propionylthiomannoside) aminopentyl) spermidine and N⁴ -(5-(α3'-propionylthiomannoside-6-phosphate) aminopentyl)spermidine, with the receptorspecific binding components included therein.

Similar results can also be obtained with N ⁴ -octylspermidine; N⁴-dodecylspermidine, fusogenic peptides acylated on the N-terminus by N⁴-(5-carboxypentyl) spermidine; N⁴ -(5-(cholest-5-en-3'-β-carbamoyl)-aminopentyl)spermidine; and N⁴-(5-(3α,7α,12αtrihydroxy-5β-cholan-24-oic) aminopentyl)-spermidineamide, with the endosome membrame disruption promoting componentincluded therein.

EXAMPLE 13 A Kit for Research and Manufacturing Use

A kit for using the multifunctional molecular complexes of the presentinvention in a research and manufacturing setting, where the individualusers supply their own DNA, includes a vial containing the transfermoiety of the present invention dissolved at 0.1 to 10 mg/mL andpreferably at 1 mg/mL in a sterile buffer at pH 6-8 and preferably at pH6.5 to 7.5. Acceptable buffers would include citrate, HEPES andphosphate. Use of the kit involves removing an aliquot of transfermoiety and adding the aliquot to the solution of DNA (at 0.05 to 2mg/mL, and preferably 0.25 to 0.75 mg/mL), such that the final ratio ofmg of transfer moiety to mg DNA is between 0.5 and 5.0 mg/mg. Optimalratios can be readily determined. The DNA-transfer moiety mixture ismixed briefly and held at 370 for 15-60 minutes. The DNA-transfer moietymixture, which has now formed the multifunctional molecular complex ofthe present invention, is then diluted with minimal essential media(serum free) to a concentration of 5 to 100 μg/mL and added to the cellsin culture. Optimal concentrations can be readily determined.

EXAMPLE 14 A Kit for Clinical and Veterinary Use

A kit for using the transfer moieties of the present invention in aclinical or veterinary setting, where the individual users supply theirown DNA, includes a vial containing the transfer moiety dissolved at 0.1to 10 mg/mL and preferably at 1 mg/mL in a sterile buffer at pH 6-8 andpreferably at pH 6.5 to 7.5. Acceptable buffers include citrate, HEPESand phosphate. Use of the kit involves removing an aliquot of transfermoiety and adding the aliquot to the solution of DNA (at 0.05 to 2mg/mL, and preferably 0.25 to 0.75 mg/mL), such that the final ratio ofmg of transfer moiety to mg DNA is between 0.5 and 5.0 mg/mg and ispreferably 1 mg/mg. Optimal ratios can be readily determined. TheDNA-compound mixture is mixed briefly and held at ambient temperaturefor 30-60 minutes. The DNA-transfer moiety mixture (10 to 500 μg ofDNA), which has now formed the multifunctional molecular complex of thepresent invention, is then injected into the patient or subject, humanor animal, as is consistent with the desired application, e.g., i.m.injection for immunization, i.v. injection for liver localization, etc.

EXAMPLE 15 A Kit for Clinical and Veterinary Use, Including DNA

A kit for using the multifunctional molecular complexes of the presentinvention in a clinical or veterinary setting, where the DNA is suppliedas part of the kit, includes a vial containing the compound dissolved at0.05 to 10 mg/mL and preferably at 0.5 to 1 mg/mL in a sterile buffer atpH 6-8, and preferably at pH 6.5 to 7.5. Acceptable buffers includecitrate, HEPES and phosphate. The kit also contains DNA appropriate forthe intended use (at 0.05 to 2 mg/μL, and preferably 0.25 to 0.75mg/mL), such that the final ratio of mg of the transfer moiety componentof the kit to mg of the DNA component of the kit, is between 0.5 and 5.0mg/mg, and is preferably 1 mg/mg. Optimal ratios can be readilydetermined. The DNA-transfer moiety mixture is held at ambienttemperature for 30-60 minutes. The DNA-transfer moiety mixture (10 to500 μg of DNA), is then injected into the patient or subject, human oranimal, as is consistent with the desired application, e.g., i.m.injection for immunization, i.v. injection for liver localization, etc.

EXAMPLE 16 Kit Containing Lyophilized Components

The kits described in Examples 13 through 15 above, can also have thecomponents thereof supplied as lyophilized powders where the transfermoieties, buffer components and excipients are reconstituted at the siteof use by the addition of sterile water. These lyophilized kits can alsoinclude the DNA as a lyophilized component.

                  TABLE 1                                                         ______________________________________                                        Picornavirus Family                                                           Genera: Rhinoviruses: (Medical) responsible for ˜ 50%                           cases of the common cold.                                                     Etheroviruses: (Medical) includes                                             polioviruses, coxsackieviruses, echoviruses,                                  and human enteroviruses such as hepatitis A                                   virus.                                                                        Apthoviruses: (Veterinary) these are the foot                                 and mouth disease viruses.                                            Target antigens: VP1, VP2, VP3, VP4, VPG                                      Calcivirus Family                                                             Genera: Norwalk Group of Viruses: (Medical) these                                     viruses are an important causative agent of                                   epidemic gastroenteritis.                                             Togavirus Family                                                              Genera: Alphaviruses: (Medical and Veterinary)                                        examples include Senilis viruses, RossRiver                                   virus and Eastern & Western Equine                                            encephalitis.                                                                 Reovirus: (Medical) Rubella virus.                                    Flariviridue Family                                                           Examples include: (Medical) dengue, yellow                                    fever, Japanese encephalitis, St. Louis                                       encephalitis and tick borne encephalitis                                      viruses.                                                                      Hepatitis C Virus: (Medical) these viruses are not placed in                  a family yet but are believed to be either a togavirus or a                   flavivirus. Most similarity is with togavirus family.                         Coronavirus Family: (Medical and Veterinary)                                  Infectious bronchitis virus (poultry)                                         Porcine transmissible gastroenteric virus                                     (pig)                                                                         Porcine hemagglutinating encephalomyelitis                                    virus (pig)                                                                   Feline infectious peritonitis virus (cats)                                    Feline enteric coronavirus (cat)                                              Canine coronavirus (dog)                                                      The human respiratory coronaviruses cause ˜40                           cases of common cold. EX. 224E, 0C43                                          Note - coronaviruses may cause non-A, B or C                                  hepatitis                                                                     Target antigens:                                                              E1 - also called M or matrix protein                                                  E2 - also called S or Spike protein                                           E3 - also called HE or hemagglutin-                                           elterose glycoprotein (not present in all                                     coronaviruses)                                                                N - nucleocapsid                                                      Rhabdovirus Family                                                            Genera: Vesiliovirus                                                                  Lyssavirus: (medical and veterinary) rabies                           Target antigen: G protein                                                             N protein                                                             Filoviridue Family: (Medical)                                                 Hemorrhagic fever viruses such as Marburg and                                 Ebola virus                                                                   Paramyxovirus Family:                                                         Genera: Paramyxovirus: (Medical and Veterinary)                                       Mumps virus, New Castle disease virus                                         (important pathogen in chickens)                                              Morbillivirus: (Medical and Veterinary)                                       Measles, canine distemper                                                     Pneuminvirus: (Medical and Veterinary)                                        Respiratory syncytial virus                                           Orthomyxovirus Family (Medical)                                               The Influenza virus                                                           Bungavirus Family                                                             Genera: Bungavirus: (Medical) California encephalitis,                                LA Crosse                                                                     Phlebovirus: (Medical) Rift Valley Fever                                      Hantavirus: Puremala is a hemahagin fever                                     virus                                                                         Nairvirus (Veterinary) Nairobi sheep disease                                  Also many unassigned bungaviruses                                     Arenavirus Family (Medical)                                                   LCM, Lassa fever virus                                                        Reovirus Family                                                               Genera: Reovirus: a possible human pathogen                                           Rotavirus: acute gastroenteritis in children                                  Orbiviruses: (Medical and Veterinary)                                         Colorado Tick fever, Lebombo (humans) equine                                  encephalosis, blue tongue                                             Retrovirus Family                                                             Sub-Family:                                                                   Oncorivirinal: (Veterinary) (Medical) feline                                  leukemia virus, HTLVI and HTLVII                                              Lentivirinal: (Medical and Veterinary) HIV,                                   feline immunodeficiency virus, equine                                         infections, anemia virus                                                      Spumavirinal                                                                  Papovavirus Family                                                            Sub-Family:                                                                   Polyomaviruses: (Medical) BKU and JCU viruses                                 Sub-Family:                                                                   Papillomavirus: (Medical) many viral types                                    associated with cancers or malignant                                          progression of papilloma                                                      Adenovirus (Medical)                                                          EX AD7, ARD., O.B. - cause respiratory disease -                              some adenoviruses such as 275 cause enteritis                                 Parvovirus Family (Veterinary)                                                Feline parvovirus: causes feline enteritis                                    Feline panleucopeniavirus                                                     Canine parvovirus                                                             Porcine parvovirus                                                            Herpesvirus Family                                                            Sub-Family: alphaherpesviridue                                                Genera: Simplexvirus (Medical)                                                        HSVI, HSVII                                                                   Varicellovirus: (Medical - Veterinary)                                        pseudorabies - varicella zoster                                       Sub-Family - betaherpesviridue                                                Genera: Cytomegalovirus (Medical)                                                     HCMV                                                                          Muromegalovirus                                                       Sub-Family: Gammaherpesviridue                                                Genera: Lymphocryptovirus (Medical)                                                   EBV - (Burkitts lympho)                                                       Rhadinovirus                                                          Poxvirus Family                                                               Sub-Family: Chordopoxviridue (Medical - Veterinary)                           Genera: Variola (Smallpox)                                                            Vaccinia (Cowpox)                                                             Parapoxivirus - Veterinary                                                    Auipoxvirus - Veterinary                                                      Capripoxvirus                                                                 Leporipoxvirus                                                                Suipoxvirus                                                           Sub-Family: Entemopoxviridue                                                  Hepadnavirus Family                                                           Hepatitis B virus                                                             Unclassified                                                                  Hepatitis delta virus                                                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Bacterial pathogens                                                           Pathogenic gram-positive cocci include: pneumococcal;                         staphylococcal; and streptococcal. Pathogenic gram-                           negative cocci include: meningococcal; and gonococcal.                        Pathogenic enteric gram-negative bacilli include:                             enterobacteriaceae; pseudomonas, acinetobacteria and                          eikenella; melioidosis; salmonella; shigellosis;                              hemophilus; chancroid; brucellosis; tularemia;                                yersinia (pasteurella); streptobacillus moniliformis                          and spirillum; listeria monocytogenes; erysipelothrix                         rhusiopathiae; diphtheria; cholera; anthrax;                                  donovanosis (granuloma inguinale); and bartonellosis.                         Pathogenic anaerobic bacteria include: tetanus;                               botulism; other clostridia; tuberculosis; leprosy; and                        other mycobacteria. Pathogenic spirochetal diseases                           include: syphilis; treponematoses: yaws, pinta and                            endemic syphilis; and leptospirosis.                                          Other infections caused by higher pathogen bacteria                           and pathogenic fungi include: actinomycosis;                                  nocardiosis; cryptococcosis, blastomycosis,                                   histoplasmosis and coccidioidomycosis; candidiasis,                           aspergillosis, and mucormycosis; sporotrichosis;                              paracoccidiodomycosis, petriellidiosis, torulopsosis,                         mycetoma and chromomycosis; and dermatophytosis.                              Rickettsial infections include rickettsial and                                rickettsioses.                                                                Examples of mycoplasma and chlamydial infections                              include: mycoplasma pneumoniae; lymphogranuloma                               venereum; psittacosis; and perinatal chlamydial                               infections.                                                                   Pathogenic eukaryotes                                                         Pathogenic protozoans and helminths and infections                            thereby include: amebiasis; malaria; leishmaniasis;                           trypanosomiasis; toxoplasmosis; pneumocystis carinii;                         babesiosis; giardiasis; trichinosis; filariasis;                              schistosomiasis; nematodes; trematodes or flukes; and                         cestode (tapeworm) infections.                                                ______________________________________                                    

What is claimed is:
 1. A multifunctional molecular complex for thetransfer of a nucleic acid composition to a target cell comprising incombination: A) said nucleic acid composition non-covalently bonded toB) a transfer moiety, wherein said transfer moiety comprises one or morecationic polyamine components bound to said nucleic acid composition,each independently comprising a cationic polyamine of the formula (1):

    NR(R.sup.3)-- --(CR.sup.1 R.sup.2).sub.m --N(R.sup.3)--!.sub.n --(CR.sup.1 R.sup.2).sub.m --NR(R.sup.3)                              (1)

wherein: R, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; m in each occurrence isindependently selected from the integers 2 through 5 inclusive; n isselected from the integers 1 through 10 inclusive; R³ is independentlyselected from the group consisting of hydrogen; C₁₋₆ alkyl; and one ormore endosome membrane disruption promoting components independentlyselected from the group consisting of:a) --B--(CR¹ R²)_(j) --C(R)₃,where R, R¹ and R² are each independently defined as above; j is aninteger from 6 to 24 inclusive; and B is optionally absent, or is abridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently defined as above; and Z is O, S, N(R), or is absent;b) --B--(R⁴)R, where R, R¹ and R² are each independently defined asabove; B cannot be absent and is a bridging group independently selectedfrom groups i) through iv) above, and the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently defined as above; X is O, S, N(R), or absent; and R⁴ isindependently selected from the group consisting of:i) fusogenicpeptides comprising spike glycoproteins of enveloped animal viruses; ii)cholic acid derivatives of the formula (2): ##STR5## where: represents abond of unspecified stereochemistry;- - - represents a single or doublebond, forming a saturated or unsaturated portion of the ring system,provided that they cannot both be unsaturated at the same time, wherebythe ring system must be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H,--C(═O)NH₂, --OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is aninteger from 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR6## where: represents a bond ofunspecified stereochemistry;- - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; PROVIDED THAT R³ is one or more endosome membrane disruptionpromoting components attached to at least one nitrogen atom of at leastone of said cationic polyamine components; and OPTIONALLY, R³ may be oneor more groups defined below, attached either to a further nitrogen atomof at least one of said cationic polyamine components to which said oneor more endosome membrane disruption promoting components is attached,or to a nitrogen atom of at least one further polyamine component whichdoes not have attached thereto any endosome membrane disruptionpromoting component: c) --B--(R⁵)R, where B cannot be absent, and is abridging group independently selected from groups i) through v)inclusive; R is independently defined as above; and R⁵ is a receptorspecific binding component independently selected from the groupconsisting of: i) D-biotin; ii) β-3'-propionylgalactosyl-β1-4-thioglucoside; iii) N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶ -bis(β1-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; v) 5-methyitetrahydrofolate; vi) folic acid;vii) folinic acid; viii) α-3'-propionyl thiomannoside; and ix)α-3'-propionyl thiomannoside-6-phosphate.
 2. A complex according toclaim 1 wherein at least one of the R³ groups thereof has the formula--B--(CR¹ R²)_(j) --C(R)₃, where B is absent; R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; and j is an integer from 6 to 24 inclusive.
 3. A complexaccording to claim 2 where R, R¹ and R² are each hydrogen, wherever theyoccur; m is 3 or 4; n is 1 to 6; and for the formula in claim 2, R, R¹and R² are each hydrogen, wherever they occur; and j is an integer offrom 8 to 18, inclusive.
 4. A complex according to claim 3 wherein j isan integer of from 8 to 12, inclusive.
 5. A complex according to claim 3wherein two of the R³ groups have the formula --B--(CR¹ R²)_(j) --C(R)₃.6. A complex according to claim 1 wherein at least one of the R³ groupsthereof has the formula --(CR¹ R²)_(j') --X(R⁴)R, where R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and j is an integer from 6 to 24 inclusive; X is N; and R⁴is independently selected from the group consisting of i) fusogenicpeptides comprising spike glycoproteins of enveloped animal viruses; andii) cholic acid and derivatives selected from the group consisting of3α, 7α, 12α-trihydroxy-5β-cholan-24-oic ester and amide.
 7. A complexaccording to claim 6 where R, R¹ and R² are each hydrogen, wherever theyoccur; m is 3 or 4; n is 1 to 6; and for the formula in claim 6, R, R¹and R² are each hydrogen, wherever they occur; j' is an integer from 2to 4 inclusive; and R⁴ is 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic esteror amide.
 8. A complex according to claim 7 wherein two of the R³ groupshave the formula --(CR¹ R²)_(j') -X(R⁴)R.
 9. A complex according toclaim 1 wherein at least one of the R³ groups thereof has the formula

    --(CR.sup.1 R.sup.2).sub.j' --X(R.sup.4)R,

where R, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; and j is an integer from 6 to 24inclusive; X is O or S; and R⁴ is independently selected from the groupconsisting of i) fusogenic peptides comprising spike glycoproteins ofenveloped animal viruses; and ii) cholesteryl and derivatives selectedfrom the group consisting of cholest-5-en-3'-β-carbonate, -β-carbamate,and -β-methylenecarboxamide.
 10. A complex according to claim 9 where R,R¹ and R² are each hydrogen, wherever they occur; m is 3 or 4; n is 1 to6; and for the formula in claim 9, R, R¹ and R² are each hydrogen,wherever they occur; j' is an integer from 2 to 4 inclusive; and R⁴ ischolest-5-en-3'-β-carbonate, -β-carbamate, or -β-methylenecarboxamide.11. A complex according to claim 10 wherein two of the R³ groups havethe formula --(CR¹ R²)_(j') --X (R⁴)R.
 12. A complex according to claim1 wherein, in addition to being an endosome membrane disruptionpromoting component as defined in b) or c) of claim 1, at least one ofthe R³ groups thereof has the formula --(CR¹ R²)_(j') --N(R⁵)R, where R,R¹ and R² are each independently defined as above; j' is an integer from6 to 24 inclusive; and R¹ is a receptor specific binding componentindependently selected from the group consisting of:i) D-biotin; ii)β-3'-propionyl galactosyl-β1-4-thioglucoside; iii) N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine; v)5-methyitetrahydrofolate; vi) folic acid; vii) folinic acid; viii)α-3'-propionyl thiomannoside; and ix) α-3'-propionylthiomannoside-6-phosphate.
 13. A complex according to claim 12 where R,R¹ and R² are each hydrogen, wherever they occur; m is 3 or 4; n is 1 to6; and for the formula in claim 12, R, R¹ and R² are each hydrogen,wherever they occur; j' is an integer from 2 to 4 inclusive; and R⁵ is areceptor specific binding component independently selected from thegroup consisting of:i) D-biotin; ii) β-3'-propionylgalactosyl-β1-4-thioglucoside; iii) N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶ -bis(β1-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; v) 5-methyitetrahydrofolate; vi) folic acid;vii) folinic acid; viii) α-3'-propionyl thiomannoside; and ix)α-3'-propionyl thiomannoside-6-phosphate.
 14. A complex according toclaim 13 wherein two of the R³ groups have the formula --(CR¹ R²)_(j')--N(R⁵)R.
 15. A complex according to claim 1 wherein R³ has the formula--B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are each hydrogen; j is aninteger from 6 to 24 inclusive; and B is a bridging group of theformula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach hydrogen; and Z is O, S, N(R), or is absent.
 16. A complexaccording to claim 1 wherein R³ has the formula --B--(R⁴)R, where R isindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; and B is a member independently selected from the groupconsisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach hydrogen; and Z is O, S, N(R), or is absent; and

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachhydrogen; X is O, S, N(R), or absent; and R⁴ is as defined in claim 1.17. A complex according to claim 1 wherein R³ has the formula--B--(R¹)R, where R is independently selected from the group consistingof hydrogen and C₁₋₆ alkyl; and B is a member independently selectedfrom the group consisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.p --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach hydrogen; and Z is O, S, N(R), or is absent; and

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachhydrogen; X is O, S, N(R), or absent; and R⁵ is as defined in claim 1.18. The multifunctional molecular complex of claim 1 wherein saidtransfer moiety comprising one or more cationic polyamine componentsbound to said nucleic acid composition, each independently comprising acationic polyamine of the formula (1):

    NR(R.sup.3)-- --(CR.sup.1 R.sup.2).sub.m --N(R.sup.3)--!.sub.n --(CR.sup.1 R.sup.2).sub.m --NR(R.sup.3)                              (1)

wherein: R, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; m in each occurrence isindependently selected from the integers 2 through 5 inclusive; n isselected from the integers 1 through 10 inclusive; R³ is independentlyselected from the group consisting of hydrogen; C₁₋₆ alkyl; and one ormore endosome membrane disruption promoting components independentlyselected from the group consisting of:a) --B--(CR¹ R²)_(j) --C(R)₃,where R, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; j is an integer from 6 to 24inclusive; and B is optionally absent, or is a bridging group of theformula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R,R¹ and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; B cannot be absent and is a bridging groupindependently selected from groups i) through iv) above, and the groupof the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) cholic acid derivatives of the formula (2):##STR7## where: represents a bond of unspecified stereochemistry;- - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or--O(CH₂ CH₂ O)_(n') H, where n' is an integer from 1 to 6 inclusive; R⁷is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and R⁸is C₁₋₆ alkyl; and ii) cholesteryl derivatives of the formula (3):##STR8## where: represents a bond of unspecified stereochemistry;- - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═C)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆ alkyl; PROVIDED THATR³ is one or more endosome membrane disruption promoting componentsattached to at least one nitrogen atom of at least one of said cationicpolyamine components.
 19. A complex according to claim 18 wherein atleast one of the R³ groups thereof has the formula --B--(CR¹ R²)_(j)--C(R)₃, where B is absent; R, R¹ and R² are selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; and j is an integer from 6 to 24inclusive.
 20. A complex according to claim 18 where R, R¹ and R² areeach hydrogen, wherever they occur; m is 3 or 4; n is 1 to 6; and j isan integer of from 8 to 18, inclusive.
 21. A complex according to claim20 wherein j is an integer of from 8 to 12, inclusive.
 22. A complexaccording to claim 20 wherein two of the R³ groups have the formula--B--(CR¹ R²)_(j) --C(R)₃.
 23. A complex according to claim 18 whereinat least one of the R³ groups thereof has the formula --(CR¹ R²)_(j')--X (R⁴)R, where R, R¹ and R² are selected from the group consisting ofhydrogen and is an integer from 1 to 8 inclusive; X is N; and R⁴ isindependently selected from the group consisting of cholic acid andderivatives selected from the group consisting of 3α, 7α,12α-trihydroxy-5β-cholan-24-oic ester and amide.
 24. A complex accordingto claim 23 where R, R¹ and R² are each hydrogen, wherever they occur; mis 3 or 4; n is 1 to 6; and for the formula in claim 6, R, R' and R ²are each hydrogen, wherever they occur; j' is an integer from 2 to 4inclusive; and R' is 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic ester oramide.
 25. A complex according to claim 24 wherein two of the R³ groupshave the formula --(CR¹ R²)_(j') --X (R⁴)R.
 26. A complex according toclaim 18 wherein at least one of the R³ groups thereof has the formula

    --(CR.sup.1 R.sup.2).sub.j' --X(R.sup.4)R,

where R, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; j' is an integer from 1 to 8inclusive; X is O or S; and R⁴ is independently selected from the groupconsisting of cholesteryl and derivatives selected from the groupconsisting of cholest-5-en-3'-β-carbonate, -β-carbamate, and-β-methylenecarboxamide.
 27. A complex according to claim 26 where R, R¹and R² are each hydrogen, wherever they occur; m is 3 or 4; n is 1 to 6;j' is an integer from 2 to 4 inclusive; and R⁴ ischolest-5-en-3'-β-carbonate, -β-carbamate, or -β-methylenecarboxamide.28. A complex according to claim 27 wherein two of the R³ groups havethe formula --(CR¹ R²)_(j') --X (R⁴)R.
 29. A self-assembling deliverysystem for the transfer of a nucleic acid composition to a target cellcomprising the following separate components capable of being broughttogether and chemically joined into a molecular complex by simplemixing: A) said nucleic acid composition to be transferred; and B) atransfer moiety comprising a cationic polyamine component which iscapable of binding to said nucleic acid composition, comprising acationic polyamine capable of being bound to said nucleic acidcomposition, comprising a cationic polyamine of formula (1) according toclaim
 1. 30. A transfer moiety capable of being brought together with anucleic acid composition and being chemically joined thereto to form amolecular complex by simple mixing, so as to form a delivery system forthe transfer of said nucleic acid composition to a target cell,comprising a cationic polyamine component which is capable of binding tosaid nucleic acid composition, comprising a cationic polyamine capableof being bound to said nucleic acid composition, comprising a cationicpolyamine of formula (1) according to claim
 1. 31. A multifunctionalmolecular complex for the transfer of a nucleic acid composition to atarget cell comprising in combination:a) said nucleic acid compositionnon-covalently bonded to b) a transfer moiety, wherein said transfermoiety comprises one or more cationic polyamine components bound to saidnucleic acid composition, each independently comprising a cationicpolyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is selected from the group consisting of one or moreendosome membrane disruption promoting components selected from thegroup consisting of:a) --B--(CR¹ R²)--C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R isselected from the group consisting of hydrogen and C₁₋₆ alkyl or absent,R¹ and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; B cannot be absent and is a bridging groupindependently selected from groups i) through iv) above, and the groupof the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula (2): ##STR9## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H,--C(═O)NH₂,--OC(═O)NH₂, --NH₂ or --O(CH₂ CH₂ O)_(n') H, where n' is aninteger from 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR10## where: represents a bond ofunspecified stereochemistry; - - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; and c) --B--(R⁵)R, where B cannot be absent, and is a bridginggroup independently selected from groups i) through v) inclusive; R isindependently selected from the group consisting of hydrogen and C₁₋₆alkyl or absent; and R⁵ is a receptor specific binding componentindependently selected from the group consisting of: i) D-biotin; ii)β-3'-propionyl galactosyl-β1-4-thioglucoside; iii) N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine; v)5-methyitetrahydrofolate; vi) folic acid; vii) folinic acid; viii)α-3'-propionyl thiomannoside; and ix) α-3'-propionylthiomannoside-6-phosphate.
 32. The multifunctional molecular complex ofclaim 31 wherein said transfer moiety comprises a cationic polyamine ofthe formula (1):

    H.sub.2 N--CH.sub.2)--N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is selected from the group consisting of C₁₋₆ alkyl; and oneor more endosome membrane disruption promoting components selected fromthe group consisting of:a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R²are each independently selected from the group consisting of hydrogenand C₁₋₆ alkyl; j is an integer from 6 to 24 inclusive; and B isoptionally absent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; and b) --B--(R⁴)R, whereR is selected from the group consisting of hydrogen and C₁₋₆ alkyl; orabsent, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; B cannot be absent and is abridging group independently selected from groups i) through iv) above,and the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula (2): ##STR11## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR12## where: represents a bond ofunspecified stereochemistry; - - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl.
 33. The multifunctional molecular complex of claim 31 whereinsaid transfer moiety comprises a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2)--NH.sub.2)(1)

wherein: R³ is --B--(CR¹ R²)--C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent.
 34. The multifunctionalmolecular complex of claim 31 wherein said transfer moiety comprises acationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2)--N(R.sup.3)--(CH.sub.2)--NH.sub.2    (1)

wherein: R³ is --B--(R⁴)R, where R is as selected from the groupconsisting of hydrogen and C₁₋₆ alkyl or absent, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; B cannot be absent and is a bridging group independently selectedfrom group consisting of

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula (2): ##STR13## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C.sub.₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR14## where: represents a bond ofunspecified stereochemistry; - - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl.
 35. The multifunctional molecular complex of claim 31 whereinsaid transfer moiety comprises a cationic polyamine selected from thegroup consisting of:N⁴ -(5- N²,N⁶ -bis(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl-N⁶ -(β-3'-propionylgalactosyl-β1-4-thioglucoside)lysyl!aminopentyl)-spermidine; N⁴-(5-cholestene-3'-oxycarbonyl)-aminopentyl)spermidine; N⁴-(5-(β-3'-propionyl galactosyl-β1-4-thioglucoside)aminopentyl)spermidine; N⁴ -(5-(methyltetrahydrofolyl)aminopentyl)-spermidine; N⁴-octylspermidine; and N⁴ -dodecylspermidine.
 36. The multifunctionalmolecular complex of claim 31 wherein said transfer moiety comprisesN⁴-(5-cholestene-3'β-oxycarbonyl)-aminopentyl)spermidine.
 37. Amultifunctional molecular complex for the transfer of a nucleic acidcomposition to a target cell comprising in combination:a) said nucleicacid composition; and b) a transfer moiety comprising one or morecationic polyamine components bound to said nucleic acid composition,each independently comprising a cationic polyamine of the formula (1):

    H.sub.2 N--CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein: R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R isselected from the group consisting of hydrogen and C₁₋₆ alkyl or absent,R¹ and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; B cannot be absent and is a bridging groupindependently selected from groups i) through iv) above, andadditionally from the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula ##STR15## where: represents a bond of unspecifiedstereochemistry;- - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR16## where: represents a bond ofunspecified stereochemistry;- - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; and c) --B--(R⁵)R, where B cannot be absent, and is a bridginggroup independently selected from groups i) through v) inclusive; R isabsent or independently selected from the group consisting of hydrogenand C₁₋₆ alkyl; and R⁵ is a receptor specific binding componentindependently selected from the group consisting of: i) D-biotin; ii)β-3'-propionyl galactosyl-β1-4-thioglucoside; iii) N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine; v)5-methyitetrahydrofolate; vi) folic acid; vii) folinic acid; viii)α-3'-propionyl thiomannoside; and ix) α-3'-propionylthiomannoside-6-phosphatePROVIDED THAT at least one of the two R³ is anendosome disruption promoting component.
 38. The multifunctionalmolecular complex of claim 37 wherein said transfer moiety comprises oneor more cationic polyamine components bound to said nucleic acidcomposition, each independently comprising a cationic polyamine of theformula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein: one of the two R³ is hydrogen and the other R³ is selected fromthe group consisting of: C₁₋₆ alkyl; and one or more endosome membranedisruption promoting components independently selected from the groupconsisting of: a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R isas selected from the group consisting of hydrogen and C₁₋₆ alkyl orabsent, R¹ and R² are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl; B cannot be absent and is abridging group independently selected from groups i) through iv) above,and the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula (2): ##STR17## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and iii) cholesterylderivatives of the formula (3): ##STR18## where: represents a bond ofunspecified stereochemistry;- - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; and c) --B--(R⁵)R, where B cannot be absent, and is a bridginggroup independently selected from groups i) through v) inclusive; R isabsent or independently selected from the group consisting of hydrogenand C₁₋₆ alkyl; and R⁵ is a receptor specific binding componentindependently selected from the group consisting of: i) D-biotin; ii)β-3'-propionyl galactosyl-β1-4-thioglucoside; iii) N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine; v)5-methyitetrahydrofolate; vi) folic acid; vii) folinic acid; viii)α-3'-propionyl thiomannoside; and ix) α-3'-propionylthiomannoside-6-phosphate.
 39. The multifunctional molecular complex ofclaim 37 wherein said transfer moiety comprises one or more cationicpolyamine components bound to said nucleic acid composition, eachindependently comprising a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2)--N(R.sup.3)--(CH.sub.2).sub.4 --N (R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                    ( 1)

wherein: said R³ groups are identical and selected from the groupconsisting of: C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R isselected from the group consisting of hydrogen and C₁₋₆ alkyl; orabsent, R¹ and R² are each independently defined as above; B cannot beabsent and is a bridging group independently selected from groups i)through iv) above, and additionally from the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) fusogenic peptides comprising spikeglycoproteins of enveloped animal viruses; ii) cholic acid derivativesof the formula (2): ##STR19## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--C(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integer from1 to 6 inclusive; R⁷ is a radical that forms the point of attachment ofthe cholic acid derivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆alkylcarbonyl--; and R⁷ is C₁₋₆ alkyl; and iii) cholesteryl derivativesof the formula (3): ##STR20## where: represents a bond of unspecifiedstereochemistry;- - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R^(6a) is a radical that forms the point ofattachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; and c) --B--(R⁵)R, where B cannot be absent, and is a bridginggroup independently selected from groups i) through v) inclusive; R isabsent or independently selected from the group consisting of hydrogenand C₁₋₆ alkyl; andR⁵ is a receptor specific binding componentindependently selected from the group consisting of: i) D-biotin; ii)β-3'-propionyl galactosyl-β1-4-thioglucoside; iii) N²,N⁶-bis(β-3'-propionyl galactosyl-β1-4-thioglucoside)lysine; iv) N²,N⁶-bis(β1-3'-propionyl galactosyl-β1-4-thioglucoside)lysyl-N⁶-(β-3'-propionyl galactosyl-β1 -4-thioglucoside)lysine; v)5-methyitetrahydrofolate; vi) folic acid; vii) folinic acid; viii)α-3'-propionyl thiomannoside; and ix) α-3'-propionylthiomannoside-6-phosphate.
 40. A multifunctional molecular complex forthe transfer of a nucleic acid composition to a target cell comprisingin combination:a) said nucleic acid composition non-covalently bonded tob) a transfer moiety, wherein said transfer moiety comprises one or morecationic polyamine components bound to said nucleic acid composition,each independently comprising a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is selected from the group consisting of one or moreendosome membrane disruption promoting components selected from thegroup consisting of:a) --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, whereR isselected from the group consisting of hydrogen and C₁₋₆ alkyl or absent,R¹ and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; B cannot be absent and is a bridging groupindependently selected from groups i) through iv) above, and the groupof the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) cholic acid derivatives of the formula (2):##STR21## where: represents a bond of unspecified stereochemistry; - - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or--O(CH₂ CH₂ O)_(n') H, where n' is an integer from 1 to 6 inclusive; R⁷is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₆ alkylcarbonyl--; and R⁸is C₁₋₆ alkyl; and ii) cholesteryl derivatives of the formula (3):##STR22## where: represents a bond of unspecified stereochemistry;- - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆ alkyl.
 41. Themultifunctional molecular complex of claim 40 wherein said transfermoiety comprises a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sub.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is --B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; j is an integer from 6 to 24 inclusive; and B is optionallyabsent, or is a bridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent.
 42. The multifunctionalmolecular complex of claim 40 wherein said transfer moiety comprises acationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is --B--(R⁴)R, whereR is as selected from the groupconsisting of hydrogen and C₁₋₆ alkyl or absent, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; B cannot be absent and is a bridging group independently selectedfrom group consisting of

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) cholic acid derivatives of the formula (2):##STR23## where: represents a bond of unspecified stereochemistry; - - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or--O(CH₂ CH₂ O)_(n') H, where n' is an integer from 1 to 6 inclusive; R⁷is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and R⁸is C₁₋₆ alkyl; and ii) cholesteryl derivatives of the formula (3):##STR24## where: represents a bond of unspecified stereochemistry; - - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆ alkyl.
 43. Themultifunctional molecular complex of claim 42 wherein said transfermoiety comprises a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein: R³ is --B--(R⁴)R, whereR is as selected from the groupconsisting of hydrogen and C₁₋₆ alkyl or absent, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; B cannot be absent and is a bridging group independently selectedfrom group consisting of

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is a cholic acid derivative ofthe formula (2): ##STR25## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl.
 44. The multifunctionalmolecular complex of claim 42 wherein said transfer moiety comprises acationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --NH.sub.2( 1)

wherein:R³ is --B--(R⁴)R, whereR is as selected from the groupconsisting of hydrogen and C₁₋₆ alkyl or absent, R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; B cannot be absent and is a bridging group independently selectedfrom group consisting of

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

wherek is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

wherej' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is a cholesteryl derivative ofthe formula (3): ##STR26## where represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R^(6a) is a radical that forms the point ofattachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl.
 45. A multifunctional molecular complex for the transfer of anucleic acid composition to a target cell comprising in combination:a)said nucleic acid composition non-covalently bonded to b) a transfermoiety, wherein said transfer moiety comprises one or more cationicpolyamine components bound to said nucleic acid composition, eachindependently comprising a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein:R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: a) --B--(CR¹ R²)--C(R)₃, where R, R¹ and R² are each independentlyselected from the group consisting of hydrogen and C₁₋₆ alkyl; j is aninteger from 6 to 24 inclusive; and B is optionally absent, or is abridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent; b) --B--(R⁴)R, where R isselected from the group consisting of hydrogen and C₁₋₆ alkyl or absent,R¹ and R² are each independently selected from the group consisting ofhydrogen and C₁₋₆ alkyl; B cannot be absent and is a bridging groupindependently selected from groups i) through iv) above, andadditionally from the group of the formula:

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N (R), or absent; and R⁴ is independently selectedfrom the group consisting of:i) cholic acid derivatives of the formula(2): ##STR27## where: represents a bond of unspecifiedstereochemistry; - - - represents a single or double bond, forming asaturated or unsaturated portion of the ring system, provided that theycannot both be unsaturated at the same time, whereby the ring systemmust be either Δ4 or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂,--OC(═O)NH₂, --NH₂, or --O(CH₂ CH₂ O)_(n') H, where n' is an integerfrom 1 to 6 inclusive; R⁷ is a radical that forms the point ofattachment of the cholic acid derivative, comprising --C₁₋₆ alkyl-- or--C₁₋₆ alkylcarbonyl--; and R⁸ is C₁₋₆ alkyl; and ii) cholesterylderivatives of the formula (3): ##STR28## where: represents a bond ofunspecified stereochemistry; - - - represents a single or double bond,forming a saturated or unsaturated portion of the ring system, providedthat they cannot both be unsaturated at the same time, whereby the ringsystem must be either Δ4 or Δ5; R^(6a) is a radical that forms the pointof attachment of the cholesteryl derivative, comprising --C₁₋₆ alkyl--,--OC(═O)--, or --OCH₂ C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆alkyl; PROVIDED THAT at least one of the two R³ is an endosomedisruption promoting component.
 46. The multifunctional molecularcomplex of claim 45 wherein said transfer moiety comprises one or morecationic polyamine components bound to said nucleic acid composition,each independently comprising a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein: R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof:--B--(CR¹ R²)_(j) --C(R)₃, where R, R¹ and R² are each independentlyselected from the group consisting of hydrogen and C₁₋₆ alkyl; j is aninteger from 6 to 24 inclusive; and B is optionally absent, or is abridging group of the formula:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent.
 47. The multifunctionalmolecular complex of claim 45 wherein said transfer moiety comprises oneor more cationic polyamine components bound to said nucleic acidcomposition, each independently comprising a cationic polyamine of theformula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein:R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: --B--(R⁴)R, where R is selected from the group consisting ofhydrogen and C₁₋₆ alkyl or absent, R¹ and R² are each independentlyselected from the group consisting of hydrogen and C₁₋₆ alkyl; B cannotbe absent and is a bridging group independently selected from groupconsisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent and

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of:i) cholic acid derivatives of the formula (2):##STR29## where: represents a bond of unspecified stereochemistry; - - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or--O(CH₂ CH₂ O)_(n') H, where n' is an integer from 1 to 6 inclusive; R⁷is a radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and R⁸is C₁₋₆ alkyl; and ii) cholesteryl derivatives of the formula (3l:##STR30## where: represents a bond of unspecified stereochemistry; - - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O) R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆ alkyl; PROVIDED THAT atleast one of the two R³ is an endosome disruption promoting component.48. The multifunctional molecular complex of claim 47 wherein saidtransfer moiety comprises one or more cationic polyamine componentsbound to said nucleic acid composition, each independently comprising acationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein: R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: --B--(R⁴)R, where R is selected from the group consisting ofhydrogen and C₁₋₆ alkyl or absent, R¹ and R² are each independentlyselected from the group consisting of hydrogen and C₁₋₆ alkyl; B cannotbe absent and is a bridging group independently selected from groupconsisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent and

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently selected fromthe group consisting of cholic acid derivatives of the formula (2):##STR31## where: represents a bond of unspecified stereochemistry;- - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R⁶ is --H, --OH, --CO₂ H, --C(═O)NH₂, --OC(═O)NH₂, --NH₂, or--O(CH₂ CH₂)_(j') H, where n' is an integer from 1 to 6 inclusive; R⁷ isa radical that forms the point of attachment of the cholic acidderivative, comprising --C₁₋₆ alkyl-- or --C₁₋₆ alkylcarbonyl--; and R⁸is C₁₋₆ alkyl.
 49. The multifunctional molecular complex of claim 47wherein said transfer moiety comprises one or more cationic polyaminecomponents bound to said nucleic acid composition, each independentlycomprising a cationic polyamine of the formula (1):

    H.sub.2 N--(CH.sub.2).sub.3 --N(R.sup.3)--(CH.sub.2).sub.4 --N(R.sup.3)--(CH.sub.2).sub.3 --NH.sub.2                 ( 1)

wherein: R³ are, independently, selected from the group consisting ofhydrogen; C₁₋₆ alkyl; and one or more endosome membrane disruptionpromoting components independently selected from the group consistingof: --B--(R⁴)R, where R is selected from the group consisting ofhydrogen and C₁₋₆ alkyl or absent, R¹ and R² are each independentlyselected from the group consisting of hydrogen and C₁₋₆ alkyl; B cannotbe absent and is a bridging group independently selected from groupconsisting of:

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--Z--;               i)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--C(═O)--Z--;         ii)

    --(CR.sup.1 R.sup.2).sub.k --N(R)--{--C(═O)--CH.sub.2 --O-- --(CH.sub.2).sub.2 --O--!.sub.1 --(CH.sub.2).sub.2 --N(R)}.sub.P --C(═O)--Z--; or                                      iii)

    --(CR.sup.1 R.sup.2).sub.k --C(═O)--{--N(R)-- --(CH.sub.2).sub.2 --O--!.sub.1 --CH.sub.2 --C(═O)}.sub.p --Z--;         iv)

where k is an integer from 1 to 6 inclusive, l is an integer from 0 to 4inclusive, and p is an integer from 1 to 3 inclusive; R, R¹ and R² areeach independently selected from the group consisting of hydrogen andC₁₋₆ alkyl; and Z is O, S, N(R), or is absent and

    --(CR.sup.1 R.sup.2).sub.j' --X--,                         v)

where j' is an integer from 1 to 8 inclusive; R¹ and R² are eachindependently selected from the group consisting of hydrogen and C₁₋₆alkyl; X is O, S, N(R), or absent; and R⁴ is independently s electedfrom the group consisting of cholesteryl derivatives of the formula (3):##STR32## where: represents a bond of unspecified stereochemistry;- - -represents a single or double bond, forming a saturated or unsaturatedportion of the ring system, provided that they cannot both beunsaturated at the same time, whereby the ring system must be either Δ4or Δ5; R^(6a) is a radical that forms the point of attachment of thecholesteryl derivative, comprising --C₁₋₆ alkyl--, --OC(═O)--, or --OCH₂C(═O)--; R^(7a) is C₁₋₆ alkyl; and R^(8a) is C₁₋₆ alkyl; PROVIDED THATat least one of the two R³ is an endosome disruption promotingcomponent.